CN103432007A - New use of tooth-related stem cells - Google Patents
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- CN103432007A CN103432007A CN2013102733426A CN201310273342A CN103432007A CN 103432007 A CN103432007 A CN 103432007A CN 2013102733426 A CN2013102733426 A CN 2013102733426A CN 201310273342 A CN201310273342 A CN 201310273342A CN 103432007 A CN103432007 A CN 103432007A
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Abstract
牙齿相关干细胞的新用途。提供了牙齿相关干细胞在制备用于预防或治疗牙齿相关疾病、免疫性疾病、自身免疫性疾病、与T淋巴细胞异常活化或升高相关的疾病、红斑狼疮或系统性红斑狼疮的产品，或者制备用于修复牙齿相关组织的产品中的新用途。还提供了包含牙齿相关干细胞的组合物以及使用牙齿相关干细胞预防或治疗疾病的方法。New uses for tooth-associated stem cells. Provided are tooth-related stem cells used in the preparation of products for preventing or treating tooth-related diseases, immune diseases, autoimmune diseases, diseases related to abnormal activation or increase of T lymphocytes, lupus erythematosus or systemic lupus erythematosus, or preparing Novel use in products for the restoration of tooth-related tissues. Compositions comprising tooth-related stem cells and methods of preventing or treating diseases using tooth-related stem cells are also provided.
Description
本申请为申请号为201080005266.5、发明名称为“牙齿相关干细胞的新用途”的发明申请的分案申请。 This application is a divisional application of the invention application with the application number 201080005266.5 and the title of the invention "New Application of Teeth Related Stem Cells". the
发明领域 field of invention
本发明涉及牙齿相关干细胞的新用途。具体地，本发明涉及牙齿相关干细胞在制备用于预防或治疗与牙齿相关的疾病或病情的产品中的用途，或者在制备用于牙齿相关组织形成或修复的产品中的用途；还涉及牙齿相关干细胞在制备用于预防或治疗免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮的产品中的用途；还涉及根据上述用途的治疗方法；还涉及包含牙齿相关干细胞的组合物。 The present invention relates to a new application of tooth-related stem cells. Specifically, the present invention relates to the use of tooth-related stem cells in the preparation of products for the prevention or treatment of diseases or conditions related to teeth, or the use of products for the formation or repair of tooth-related tissues; Stem cells are used in the prevention or treatment of immune diseases or conditions, autoimmune diseases or conditions, diseases or conditions related to abnormal activation of T lymphocytes, diseases or conditions related to abnormal increase of T lymphocytes, lupus erythematosus, or Use in a product for systemic lupus erythematosus; also to a method of treatment according to the above use; also to a composition comprising tooth-associated stem cells. the
背景技术 Background technique
干细胞是一类具有自我更新和分化潜能的原始细胞，可分为胚胎干细胞和成体干细胞。在成体组织及器官中普遍存在着特异的成体干细胞，目前已经发现的例如来源于人的牙齿相关干细胞都是来源于中胚层的间充质干细胞(mesenchymal stem cells,MSCs)，包括牙髓干细胞(dental pulp stem cells，DPSCs)、脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)、牙周膜干细胞(periodontal ligament stem cells，PDLSCs)和根尖牙乳头干细胞(stem cells from apical papilla，SCAP)(1-4，即指文献1至4，下同)，它们可以来源于多种动物(例如哺乳动物，例如人)。研究表明牙齿相关干细胞具有很高的增殖能力和多向分化潜能，能够向成骨细胞、脂肪细胞、神经样细胞分化。当把牙齿相关干细胞和三维支架材料的复合物植入到裸鼠皮下时，能够产生自体牙髓牙本质复合体样结构和牙骨质牙周膜复合体样结构(1,3)。目前在自体小型猪模型上已经应用PDLSCs和SCAP成功再生出生物牙根(4)。目前国际上干细胞研究焦点之一集中在建立各种组织成体干细胞库，进行干细 胞的临床试验研究。
Stem cells are a kind of primitive cells with self-renewal and differentiation potential, which can be divided into embryonic stem cells and adult stem cells. Specific adult stem cells are ubiquitous in adult tissues and organs. The tooth-related stem cells that have been found so far are derived from mesoderm mesenchymal stem cells (mesenchymal stem cells, MSCs), including dental pulp stem cells ( dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (stem cells from apical papilla, SCAP) (1-4, referring to
根据WHO(2003)统计数字表明，牙齿相关疾病所波及的人群范围已远远超过其它任何一种疾病。目前，全世界每年用于牙齿疾病的治疗费用已超过200亿美元。在牙齿相关疾病中，牙周病是一种全世界范围内发病率很高的细菌感染性疾病，最终导致牙齿支持组织丧失和牙齿缺失，并能引起一系列的全身系统性疾病(13)；而牙齿缺失可对人体消化系统和全身健康造成严重的影响，同时也会使患者产生不良的社会心理影响。但目前对牙周病及牙齿组织缺损没有好的药物或治疗方法进行治疗或修复；有关牙齿缺失的治疗主要仍是采用人工材料制作牙齿的替代物。 According to WHO (2003) statistics show that the range of people affected by dental-related diseases has far exceeded any other disease. At present, the annual treatment cost for dental diseases in the world has exceeded 20 billion US dollars. Among tooth-related diseases, periodontal disease is a bacterial infectious disease with a high incidence worldwide, which eventually leads to the loss of tooth supporting tissue and tooth loss, and can cause a series of systemic diseases (13); Tooth loss can have a serious impact on the human digestive system and general health, and it can also cause adverse social and psychological effects on patients. But at present, there is no good medicine or treatment method for periodontal disease and tooth tissue defect to treat or repair; the treatment of tooth loss mainly still adopts artificial materials to make tooth substitutes. the
系统性红斑狼疮(systemic lupus erythematosus，SLE)是以多脏器受累和血中存在多种抗体为特征的自身免疫病，常规的免疫抑制或免疫调节治疗可使多数患者的生存期延长，生活质量得到改善。1996年意大利学者Marmont首先报道用自体骨髓移植治疗重症红斑狼疮取得成功后,国内外迅速开展了自体外周血干细胞移植和自体骨髓移植治疗重症SLE等多种难治性免疫病，取得了较好的疗效。造血干细胞移植能使部分患者达到病情控制，但其费用高、并发症多，病情复发率高达40%-50%，不能被常规使用；并且，移植后虽然部分患者有可能达到长期缓解，但均不能彻底根治自身免疫性疾病，部分患者存在复发的问题。 Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the involvement of multiple organs and the presence of multiple antibodies in the blood. Conventional immunosuppressive or immunomodulatory therapy can prolong the survival of most patients and improve the quality of life. Improved. In 1996, the Italian scholar Marmont first reported the success of autologous bone marrow transplantation in the treatment of severe lupus erythematosus. At home and abroad, autologous peripheral blood stem cell transplantation and autologous bone marrow transplantation were rapidly carried out in the treatment of severe SLE and other refractory immune diseases, and achieved good results. curative effect. Hematopoietic stem cell transplantation can make some patients achieve disease control, but it is expensive, has many complications, and the recurrence rate of the disease is as high as 40%-50%, so it cannot be used routinely; and although some patients may achieve long-term remission after transplantation, all Autoimmune diseases cannot be completely cured, and some patients may relapse. the
因此，目前仍然需要可有效治疗某些牙齿相关疾病和某些免疫性疾病的新颖方法。 Accordingly, there remains a need for novel approaches that are effective in the treatment of certain dental-related diseases and certain immune diseases. the
发明内容 Contents of the invention
本发明的目的是提供有效治疗某些牙齿相关疾病和某些免疫性疾病的新颖方法。本发明人在研究中现出人意料地发现牙齿相关干细胞例如牙周膜干细胞(PDLSCs)不但在自体的牙齿中使缺损的牙齿组织再生，而且能在异体缺损的牙齿组织使牙齿组织再生，同时PDLSCs还对异常升高的T淋巴细胞有抑制作用。本发明基于上述研究结果得以完成。 It is an object of the present invention to provide novel methods of effective treatment of certain dental-related diseases and certain immune diseases. The inventors found that tooth-related stem cells such as periodontal ligament stem cells (PDLSCs) can not only regenerate defective tooth tissue in autologous teeth, but also regenerate tooth tissue in allogeneic tooth tissue. It has an inhibitory effect on abnormally elevated T lymphocytes. The present invention has been accomplished based on the above research results. the
发明概述Summary of the invention
本发明首先涉及PDLSCs在制备用于预防或治疗牙周病，牙齿缺损修复的产品中用途。 The present invention firstly relates to the use of PDLSCs in the preparation of products for preventing or treating periodontal disease and repairing tooth defects. the
本发明还涉及PDLSCs在制备用于预防或治疗与T淋巴细胞异常升高有关的疾病或症状的产品中用途。 The present invention also relates to the use of PDLSCs in the preparation of products for preventing or treating diseases or symptoms related to the abnormal increase of T lymphocytes. the
本发明还涉及一种预防或治疗牙周病，修复缺损牙齿组织的方法，其包括给予需预防或治疗牙周病或需修复缺损牙齿组织的宿主以有效预防或治疗量或修复缺损牙齿组织量的PDLSCs。 The present invention also relates to a method for preventing or treating periodontal disease and repairing defective tooth tissue, which comprises administering an effective preventive or therapeutic amount or an amount for repairing defective tooth tissue to a host that needs to prevent or treat periodontal disease or repair defective tooth tissue PDLSCs. the
本发明涉及一种预防或治疗与T淋巴细胞异常升高有关的疾病或症状的方法，其包括给予T淋巴细胞异常升高的宿主预防或治疗有效量的PDLSCs。 The present invention relates to a method for preventing or treating diseases or symptoms associated with abnormally elevated T lymphocytes, which comprises administering a preventive or therapeutically effective amount of PDLSCs to a host with abnormally elevated T lymphocytes. the
发明详述Detailed description of the invention
更详细地，本发明提供了以下的各个方面以及各个方面的具体项。 In more detail, the present invention provides the following aspects and specific items of each aspect. the
本发明第一方面提供了牙齿相关干细胞在制备用于预防或治疗与牙齿相关的疾病或病情的产品中的用途，或者在制备用于牙齿相关组织形成或修复的产品中的用途。 The first aspect of the present invention provides the use of tooth-related stem cells in the preparation of products for the prevention or treatment of tooth-related diseases or conditions, or the use in the preparation of products for the formation or repair of tooth-related tissues. the
根据本发明第一方面任一项的用途，其中所述的牙齿相关干细胞选自：牙髓干细胞、脱落乳牙牙髓干细胞、牙周膜干细胞、和根尖牙乳头干细胞。 According to the use according to any one of the first aspect of the present invention, the tooth-related stem cells are selected from the group consisting of dental pulp stem cells, dental pulp stem cells of exfoliated deciduous teeth, periodontal ligament stem cells, and apical tooth papilla stem cells. the
根据本发明第一方面任一项的用途，其中所述的牙齿相关干细胞来自哺乳动物。在一个实施方案中，所述的牙齿相关干细胞来自选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The use according to any one of the first aspect of the present invention, wherein the tooth-related stem cells are from mammals. In one embodiment, the tooth-related stem cells are from mammals selected from the following: humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, Sheep, goats. the
根据本发明第一方面任一项的用途，其中所述的牙齿相关干细胞来自哺乳动物，并且选自：牙髓干细胞(dental pulp stem cells，DPSCs)、脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)、牙周膜干细胞(periodontal ligament stem cells，PDLSCs)和根尖牙乳头干细胞(stem cells from apical papilla，SCAP)。 According to the use of any one of the first aspect of the present invention, wherein the tooth-related stem cells are from mammals, and are selected from: dental pulp stem cells (dental pulp stem cells, DPSCs), exfoliated deciduous teeth dental pulp stem cells (stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAP). the
根据本发明第一方面任一项的用途，其中所述的与牙齿相关的疾病或病情或者牙齿相关组织形成或修复选自：牙周病、牙周炎、牙齿缺损、牙齿组织缺损、牙齿缺损修复、牙齿相关组织缺损和修复、牙齿相关组织替代物再生、等等。 Use according to any one of the first aspect of the present invention, wherein the tooth-related disease or disease or tooth-related tissue formation or repair is selected from: periodontal disease, periodontitis, tooth defect, tooth tissue defect, tooth defect Restoration, tooth-related tissue loss and restoration, regeneration of tooth-related tissue substitutes, etc. the
根据本发明第一方面任一项的用途，其中所述的牙齿相关干细胞用于自体或者异体的牙齿相关的疾病或病情，或者用于自体或异体的牙齿相关组织形成或修复。 The use according to any one of the first aspect of the present invention, wherein the tooth-related stem cells are used for autologous or allogeneic tooth-related diseases or conditions, or for autologous or allogeneic tooth-related tissue formation or repair. the
根据本发明第一方面任一项的用途，其中所述的牙齿相关组织包括但不限于牙齿、牙根、牙髓、牙龈等等，以及其它与牙齿内部、牙齿表层、 牙齿表面或者牙齿周围相关的组织。 According to the use of any one of the first aspect of the present invention, wherein the tooth-related tissues include but not limited to teeth, tooth roots, pulp, gums, etc., and other tissues related to the interior of the tooth, the surface of the tooth, the surface of the tooth or around the tooth organize. the
本发明第一方面及其各子项的特征和优点同样适用于本发明其它任一方面及其各子项。 The features and advantages of the first aspect of the invention and its sub-items are equally applicable to any other aspect of the invention and its sub-items. the
本发明第二方面提供了牙齿相关干细胞在制备用于预防或治疗免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮的产品中的用途。 The second aspect of the present invention provides tooth-related stem cells for the prevention or treatment of immune diseases or conditions, autoimmune diseases or conditions, diseases or conditions related to abnormal activation of T lymphocytes, and abnormal increase of T lymphocytes. diseases or conditions, lupus erythematosus, or systemic lupus erythematosus products. the
根据本发明第二方面任一项的用途，其中所述的牙齿相关干细胞选自：牙髓干细胞、脱落乳牙牙髓干细胞、牙周膜干细胞、和根尖牙乳头干细胞。 According to the use according to any one of the second aspect of the present invention, the tooth-related stem cells are selected from the group consisting of dental pulp stem cells, dental pulp stem cells of exfoliated deciduous teeth, periodontal ligament stem cells, and apical tooth papilla stem cells. the
根据本发明第二方面任一项的用途，其中所述的牙齿相关干细胞来自哺乳动物。在一个实施方案中，所述的牙齿相关干细胞来自选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The use according to any one of the second aspect of the present invention, wherein the tooth-related stem cells are from mammals. In one embodiment, the tooth-related stem cells are from mammals selected from the following: humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, Sheep, goats. the
根据本发明第二方面任一项的用途，其中所述的牙齿相关干细胞来自哺乳动物，并且选自：牙髓干细胞(dental pulp stem cells，DPSCs)、脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)、牙周膜干细胞(periodontal ligament stem cells，PDLSCs)和根尖牙乳头干细胞(stem cells from apical papilla，SCAP)。 According to the use of any one of the second aspect of the present invention, wherein the tooth-related stem cells are from mammals, and are selected from: dental pulp stem cells (dental pulp stem cells, DPSCs), exfoliated deciduous teeth dental pulp stem cells (stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAP). the
根据本发明第二方面任一项的用途，其中所述的牙齿相关干细胞用于自体或者异体的免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮。 According to the use of any one of the second aspect of the present invention, wherein the tooth-related stem cells are used for autologous or allogeneic immune diseases or conditions, autoimmune diseases or conditions, diseases or conditions related to abnormal activation of T lymphocytes, Diseases or conditions associated with an abnormal increase in T lymphocytes, lupus erythematosus, or systemic lupus erythematosus. the
本发明第二方面及其各子项的特征和优点同样适用于本发明其它任一方面及其各子项。 The features and advantages of the second aspect of the invention and its sub-items are equally applicable to any other aspect of the invention and its sub-items. the
本发明第三方面提供了预防或治疗与牙齿相关的疾病或病情的方法，或者形成或修复牙齿相关组织的方法，该方法包括给有需要预防或治疗与牙齿相关的疾病或病情的宿主施用有效量的牙齿相关干细胞，或者该方法包括给有需要形成或修复牙齿相关组织的宿主施用有效量的牙齿相关干细胞。 The third aspect of the present invention provides a method for preventing or treating a tooth-related disease or condition, or a method for forming or repairing a tooth-related tissue, the method comprising administering effective amount of tooth-related stem cells, or the method includes administering an effective amount of tooth-related stem cells to a host in need of forming or repairing tooth-related tissues. the
根据本发明第三方面任一项的方法，其中所述的牙齿相关干细胞选自：牙髓干细胞、脱落乳牙牙髓干细胞、牙周膜干细胞、和根尖牙乳头 干细胞。 The method according to any one of the third aspect of the present invention, wherein said tooth-related stem cells are selected from the group consisting of dental pulp stem cells, exfoliated deciduous tooth pulp stem cells, periodontal ligament stem cells, and apical tooth papilla stem cells. the
根据本发明第三方面任一项的方法，其中所述的牙齿相关干细胞来自哺乳动物。在一个实施方案中，所述的牙齿相关干细胞来自选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The method according to any one of the third aspect of the present invention, wherein said tooth-related stem cells are from mammals. In one embodiment, the tooth-related stem cells are from mammals selected from the following: humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, Sheep, goats. the
根据本发明第三方面任一项的方法，其中所述的牙齿相关干细胞来自哺乳动物，并且选自：牙髓干细胞(dental pulp stem cells，DPSCs)、脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)、牙周膜干细胞(periodontal ligament stem cells，PDLSCs)和根尖牙乳头干细胞(stem cells from apical papilla，SCAP)。 According to the method according to any one of the third aspect of the present invention, wherein said tooth-related stem cells are from mammals, and are selected from the group consisting of: dental pulp stem cells (dental pulp stem cells, DPSCs), exfoliated deciduous teeth dental pulp stem cells (stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAP). the
根据本发明第三方面任一项的方法，其中所述的与牙齿相关的疾病或病情或者牙齿相关组织形成或修复选自：牙周病、牙周炎、牙齿缺损、牙齿组织缺损、牙齿缺损修复、牙齿相关组织缺损和修复、牙齿相关组织替代物再生、等等。 According to the method according to any one of the third aspect of the present invention, wherein said tooth-related disease or condition or tooth-related tissue formation or repair is selected from: periodontal disease, periodontitis, tooth defect, tooth tissue defect, tooth defect Restoration, tooth-related tissue loss and restoration, regeneration of tooth-related tissue substitutes, etc. the
根据本发明第三方面任一项的方法，其中所述的牙齿相关干细胞用于自体或者异体的牙齿相关的疾病或病情，或者用于自体或异体的牙齿相关组织形成或修复。 The method according to any one of the third aspect of the present invention, wherein the tooth-related stem cells are used for autologous or allogeneic tooth-related diseases or conditions, or for autologous or allogeneic tooth-related tissue formation or repair. the
根据本发明第三方面任一项的方法，其中所述的牙齿相关组织包括但不限于牙齿、牙根、牙髓、牙龈等等，以及其它与牙齿内部、牙齿表层、牙齿表面或者牙齿周围相关的组织。 According to the method according to any one of the third aspect of the present invention, wherein said tooth-related tissues include but not limited to teeth, tooth roots, pulp, gums, etc. organize. the
根据本发明第三方面任一项的方法，其中所述的宿主是哺乳动物。在一个实施方案中，所述的宿主是选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The method according to any one of the third aspects of the present invention, wherein said host is a mammal. In one embodiment, the host is a mammal selected from the group consisting of humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, sheep, goat. the
在本发明第三方面任一项方法的一个实施方案中，其包括以下步骤： In one embodiment of any method of the third aspect of the present invention, it comprises the following steps:
(a)采集人牙周膜； (a) collection of human periodontal ligament;
(b)培养hPDLSCs； (b) culture hPDLSCs;
(c)任选地冷冻保存hPDLSCs； (c) optionally cryopreserving hPDLSCs;
(d)任选地融化hPDLSCs，以及任选地对hPDLSCs检查支原体、细菌、集落形成效率、间质干细胞标记模式和核型分析； (d) optionally thawing the hPDLSCs, and optionally checking the hPDLSCs for mycoplasma, bacteria, colony formation efficiency, mesenchymal stem cell marker pattern and karyotype analysis;
(e)制备hPDLSCs片； (e) prepare hPDLSCs sheet;
(f)将HA/TCP置于容纳有hPDLSCs片的器皿中； (f) HA/TCP is placed in the vessel containing hPDLSCs sheet;
(j)在任选的牙周初始治疗后，将hPDLSCs片与HA/TCP植入到牙周损伤缺陷处； (j) After the optional periodontal initial treatment, implant hPDLSCs sheets with HA/TCP into the periodontal lesion defect;
(k)任选的进行临床和放射照相评价、血液学和免疫学评价。 (k) Optionally perform clinical and radiographic evaluations, hematological and immunological evaluations. the
本发明第三方面及其各子项的特征和优点同样适用于本发明其它任一方面及其各子项。 The features and advantages of the third aspect of the present invention and its sub-items are also applicable to any other aspect of the present invention and its sub-items. the
本发明第四方面提供了预防或治疗免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮的方法，该方法包括给有此需要的宿主施用有效量的牙齿相关干细胞。 The fourth aspect of the present invention provides prevention or treatment of immune diseases or conditions, autoimmune diseases or conditions, diseases or conditions related to abnormal activation of T lymphocytes, diseases or conditions related to abnormal increase of T lymphocytes, lupus erythematosus , or a method for systemic lupus erythematosus, the method comprising administering an effective amount of tooth-associated stem cells to a host in need thereof. the
根据本发明第四方面任一项的方法，其中所述的牙齿相关干细胞选自：牙髓干细胞、脱落乳牙牙髓干细胞、牙周膜干细胞、和根尖牙乳头干细胞。 The method according to any one of the fourth aspect of the present invention, wherein said tooth-related stem cells are selected from the group consisting of dental pulp stem cells, exfoliated deciduous tooth pulp stem cells, periodontal ligament stem cells, and apical papilla stem cells. the
根据本发明第四方面任一项的方法，其中所述的牙齿相关干细胞来自哺乳动物。在一个实施方案中，所述的牙齿相关干细胞来自选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The method according to any one of the fourth aspect of the present invention, wherein said tooth-related stem cells are from mammals. In one embodiment, the tooth-related stem cells are from mammals selected from the following: humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, Sheep, goats. the
根据本发明第四方面任一项的方法，其中所述的牙齿相关干细胞来自哺乳动物，并且选自：牙髓干细胞(dental pulp stem cells，DPSCs)、脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)、牙周膜干细胞(periodontal ligament stem cells，PDLSCs)和根尖牙乳头干细胞(stem cells from apical papilla，SCAP)。 According to the method according to any one of the fourth aspect of the present invention, wherein said tooth-related stem cells are from mammals, and are selected from the group consisting of: dental pulp stem cells (dental pulp stem cells, DPSCs), exfoliated deciduous teeth dental pulp stem cells (stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAP). the
根据本发明第四方面任一项的方法，其中所述的牙齿相关干细胞用于自体或者异体的免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮。 The method according to any one of the fourth aspect of the present invention, wherein the tooth-related stem cells are used for autologous or allogeneic immune diseases or conditions, autoimmune diseases or conditions, diseases or conditions related to abnormal activation of T lymphocytes, Diseases or conditions associated with an abnormal increase in T lymphocytes, lupus erythematosus, or systemic lupus erythematosus. the
根据本发明第四方面任一项的方法，其中所述的宿主是哺乳动物。在一个实施方案中，所述的宿主是选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The method according to any one of the fourth aspects of the present invention, wherein said host is a mammal. In one embodiment, the host is a mammal selected from the group consisting of humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, sheep, goat. the
本发明第四方面及其各子项的特征和优点同样适用于本发明其它任一方面及其各子项。 The features and advantages of the fourth aspect of the present invention and its sub-items are also applicable to any other aspect of the present invention and its sub-items. the
本发明第五方面提供了一种组合物，其包含有效量的牙齿相关干细胞和任选的药学可接受的载体。 The fifth aspect of the present invention provides a composition comprising an effective amount of tooth-related stem cells and optionally a pharmaceutically acceptable carrier. the
根据本发明第五方面任一项的组合物，其中所述的牙齿相关干细胞选自：牙髓干细胞、脱落乳牙牙髓干细胞、牙周膜干细胞、和根尖牙乳头干细胞。 The composition according to any one of the fifth aspect of the present invention, wherein the tooth-related stem cells are selected from the group consisting of dental pulp stem cells, exfoliated deciduous tooth pulp stem cells, periodontal ligament stem cells, and apical papilla stem cells. the
根据本发明第五方面任一项的组合物，其中所述的牙齿相关干细胞来自哺乳动物。在一个实施方案中，所述的牙齿相关干细胞来自选自以下的哺乳动物：人、猪(例如五指山小型猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 The composition according to any one of the fifth aspect of the present invention, wherein said tooth-related stem cells are from mammals. In one embodiment, the tooth-related stem cells are from mammals selected from the following: humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cows, horses, monkeys, rats, mice, guinea pigs, sheep, Sheep, goats. the
根据本发明第五方面任一项的组合物，其中所述的牙齿相关干细胞来自哺乳动物，并且选自：牙髓干细胞(dental pulp stem cells，DPSCs)、脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth，SHED)、牙周膜干细胞(periodontal ligament stem cells，PDLSCs)和根尖牙乳头干细胞(stem cells from apical papilla，SCAP)。 The composition according to any one of the fifth aspect of the present invention, wherein the tooth-related stem cells are from mammals, and are selected from the group consisting of: dental pulp stem cells (dental pulp stem cells, DPSCs), exfoliated deciduous tooth pulp stem cells (stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAP). the
根据本发明第五方面任一项的组合物，其是用于预防或治疗与牙齿相关的疾病或病情，或者用于牙齿相关组织形成或修复，或者用于预防或治疗免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮。 The composition according to any one of the fifth aspect of the present invention, which is used for preventing or treating diseases or conditions related to teeth, or for forming or repairing tooth-related tissues, or for preventing or treating immune diseases or conditions, Autoimmune disease or condition, disease or condition related to abnormal activation of T lymphocytes, disease or condition related to abnormal increase of T lymphocytes, lupus erythematosus, or systemic lupus erythematosus. the
根据本发明第五方面任一项的组合物，其中所述的有效量是可以有效地用于预防或治疗与牙齿相关的疾病或病情的剂量，或者是要以有效地用于牙齿相关组织形成或修复的剂量，或者是要以有效地用于预防或治疗免疫性疾病或病情、自身免疫性疾病或病情、与T淋巴细胞异常活化有关的疾病或病情、与T淋巴细胞异常升高有关的疾病或病情、红斑狼疮、或者系统性红斑狼疮的剂量。 The composition according to any one of the fifth aspect of the present invention, wherein the effective amount is a dose that can be effectively used for preventing or treating tooth-related diseases or conditions, or is to be effectively used for tooth-related tissue formation or repairing dosage, or to be effective for the prevention or treatment of immune diseases or conditions, autoimmune diseases or conditions, diseases or conditions related to abnormal activation of T lymphocytes, and abnormal increase of T lymphocytes Disease or condition, lupus, or systemic lupus erythematosus. the
本发明第五方面及其各子项的特征和优点同样适用于本发明其它任一方面及其各子项。 The features and advantages of the fifth aspect of the present invention and its sub-items are also applicable to any other aspect of the present invention and its sub-items. the
根据本发明上下文的详细记载，本发明令人满意地实现上上述的各个方面及其各子项。 According to the detailed description of the context of the invention, the invention satisfactorily realizes the above-mentioned aspects and sub-items thereof. the
下面对本发明的各个方面和特点作进一步的描述。 Various aspects and features of the present invention are further described below. the
本发明所引述的所有文献，它们的全部内容通过引用并入本文，并且如果这些文献所表达的含义与本发明不一致时，以本发明的表述为准。此 外，本发明使用的各种术语和短语具有本领域技术人员公知的一般含义，即便如此，本发明仍然希望在此对这些术语和短语作更详尽的说明和解释，提及的术语和短语如有与公知含义不一致的，以本发明所表述的含义为准。 All the documents cited in the present invention are incorporated herein by reference in their entirety, and if the meaning expressed in these documents is inconsistent with the present invention, the expression of the present invention shall prevail. In addition, various terms and phrases used in the present invention have common meanings known to those skilled in the art. Even so, the present invention still hopes to make a more detailed description and explanation of these terms and phrases here. The terms and phrases mentioned If there is any inconsistency with the known meaning, the meaning expressed in the present invention shall prevail. the
本发明人在研究中现出人意料地发现PDLSCs等牙齿相关的干细胞不但在自体的牙齿中使缺损的牙齿组织再生，而且能在异体缺损的牙齿组织使牙齿组织再生。本发明基于上述研究结果得以完成。因此，本发明涉及牙齿相关干细胞例如PDLSCs在制备用于预防或治疗牙周病，牙齿缺损修复的产品中用途(例如图8所示)；还涉及一种预防或治疗牙周病，修复缺损牙齿组织的方法，其包括给予需预防或治疗牙周病或需修复缺损牙齿组织的宿主以有效预防或治疗量或修复缺损牙齿组织量的牙齿相关干细胞例如PDLSCs。 In the research, the inventors found that tooth-related stem cells such as PDLSCs can not only regenerate defective tooth tissue in autologous teeth, but also regenerate tooth tissue in allogeneic tooth tissue. The present invention has been accomplished based on the above research results. Therefore, the present invention relates to the use of tooth-related stem cells such as PDLSCs in the preparation of products for the prevention or treatment of periodontal disease and the restoration of tooth defects (such as shown in Figure 8); A method for tissue, which includes administering tooth-related stem cells such as PDLSCs to a host that needs to prevent or treat periodontal disease or repair defective tooth tissue, with an amount effective for preventing or treating or repairing defective tooth tissue. the
此外，本发明提出生物牙根再生的新理念；利用自体异体SCAP/DPSCs和PDLSCs再生出了具有生物学功能的生物牙根，在新形成的功能性生物牙根上再进行冠的修复，恢复患者的咀嚼功能(例如图10所示)；提供生物牙根再生的具体实施方法，为进一步对生物牙根再生的机理进行深入研究及生物牙根的商品化提供依据。因此，本发明涉及生物牙根再生的新理念，并涉及SCAP，DPSCs，PDLSCs等牙齿相关的干细胞在生物牙根再生中的用途。本发明还涉及生物牙根再生的具体实施办法，其中包括牙周膜干细胞膜片的制备及其所需最佳细胞数及最佳生长时间。 In addition, the present invention proposes a new concept of biological tooth root regeneration; using autologous allogeneic SCAP/DPSCs and PDLSCs to regenerate a biological tooth root with biological functions, and then restore the crown on the newly formed functional biological tooth root to restore the patient's mastication Function (as shown in Fig. 10 for example): provide a specific implementation method of biological tooth root regeneration, and provide a basis for further in-depth research on the mechanism of biological tooth root regeneration and commercialization of biological tooth root. Therefore, the present invention relates to a new concept of biological tooth root regeneration, and relates to the use of tooth-related stem cells such as SCAP, DPSCs, PDLSCs, etc. in biological tooth root regeneration. The invention also relates to a specific implementation method of biological tooth root regeneration, which includes the preparation of the periodontal ligament stem cell membrane and its required optimal cell number and optimal growth time. the
再者，本发明涉及牙齿相关干细胞例如PDLSCs在制备用于预防或治疗与T淋巴细胞异常升高有关的疾病或症状的产品中用途。本发明还涉及一种预防或治疗与T淋巴细胞异常升高有关的疾病或症状的方法，其包括给予T淋巴细胞异常升高的宿主预防或治疗有效量的牙齿相关干细胞例如PDLSCs。本发明进一步涉及牙齿相关干细胞例如SHED在预防或治疗系统性红斑狼疮病等自身免疫系统病中用途(例如图11所示)。 Furthermore, the present invention relates to the use of tooth-related stem cells such as PDLSCs in the preparation of products for preventing or treating diseases or symptoms associated with abnormally elevated T lymphocytes. The present invention also relates to a method for preventing or treating diseases or symptoms associated with abnormally elevated T lymphocytes, which comprises administering a preventive or therapeutically effective amount of tooth-related stem cells such as PDLSCs to a host with abnormally elevated T lymphocytes. The present invention further relates to the use of tooth-related stem cells such as SHED in the prevention or treatment of autoimmune diseases such as systemic lupus erythematosus (for example, as shown in FIG. 11 ). the
根据本发明，术语“牙周病”包括但不限于牙周炎。 According to the present invention, the term "periodontal disease" includes but is not limited to periodontitis. the
根据本发明，术语“缺损牙齿组织”包括但不限于宿主牙齿的各种缺损情况，如各种原因引起的牙齿脱落。 According to the present invention, the term "defective tooth tissue" includes but not limited to various defect situations of host teeth, such as tooth loss caused by various reasons. the
根据本发明，术语“宿主”通常指哺乳动物，包括但不限于人，猪，牛，马等。在一个实施方案中，术语“宿主”是指人、猪(例如五指山小型 猪、贵州香猪)、牛、马、猴、大鼠、小鼠、豚鼠、羊、绵羊、山羊。 According to the present invention, the term "host" generally refers to mammals, including but not limited to humans, pigs, cows, horses and the like. In one embodiment, the term "host" refers to humans, pigs (such as Wuzhishan miniature pigs, Guizhou fragrant pigs), cattle, horses, monkeys, rats, mice, guinea pigs, sheep, sheep, goats. the
根据本发明，术语“产品”是指适于PDLSCs应用的各种形式。根据本发明，术语“产品”还指适于牙齿相关干细胞应用的各种形式，例如组合物、药物组合物等。 According to the present invention, the term "product" refers to various forms suitable for the application of PDLSCs. According to the present invention, the term "product" also refers to various forms suitable for the application of tooth-related stem cells, such as compositions, pharmaceutical compositions and the like. the
根据本发明，术语“与牙齿相关的疾病或病情”是指本文所述宿主罹患或者表现的疾病、病情、体症、身体状态等，这些疾病、病情、体症、身体状态等与牙齿相关。 According to the present invention, the term "diseases or conditions related to teeth" refers to diseases, conditions, symptoms, physical conditions, etc. that the host described herein suffers from or exhibits, and these diseases, conditions, symptoms, physical conditions, etc. are related to teeth. the
根据本发明，术语“牙齿相关组织形成或修复”或者术语“牙齿相关组织的形成或修复”或者“形成或修复牙齿相关组织”等，它们具有相同或近似的含义，并且通常是指本文所述宿主的牙齿相关组织进行形成、修复、生成、再生、培养等处理或操作，或者对牙齿相关组织异常(例如缺损)进行形成、修复、生成、再生、培养等处理或操作。 According to the present invention, the term "tooth-related tissue formation or repair" or the term "tooth-related tissue formation or repair" or "tooth-related tissue formation or repair", etc., have the same or similar meanings, and generally refer to the The host tooth-related tissue is formed, repaired, generated, regenerated, and cultivated, or the tooth-related tissue abnormality (such as a defect) is formed, repaired, generated, regenerated, and cultivated. the
根据本发明，术语“组合物”是具有本领域技术人员通常理解的含义，并且通常是指可接或间接(例如临用前稀释)用于临床使用的形式，例如剂型、药物剂型、给药形式、等。在临床应用领域或者药物领域，术语“组合物”还通常与“药物组合物”具有同等含义。 According to the present invention, the term "composition" has the meaning generally understood by those skilled in the art, and generally refers to a form that can be used directly or indirectly (such as dilution before use) for clinical use, such as dosage form, pharmaceutical dosage form, administration form, etc. In the field of clinical application or medicine, the term "composition" usually has the same meaning as "pharmaceutical composition". the
可改变本发明组合物或药物组合物中牙齿相关干细胞的实际剂量水平，以便所得的干细胞量能有效针对具体宿主、患者在特定组合物及其组成以及相应给药方式的情况下得到所需的治疗或预防反应。剂量水平须根据具体干细胞的活性、给药途径、所治疗病况的严重程度、疾病或病情的治疗进程、形成和修复(以及生成、再生、培养等)处理或操作的进程、以及待治疗患者的病况和既往病史来选定。但是，本领域的做法是，干细胞的剂量以及施用时间从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。因此，就本发明而言，本领域技术人员在本发明详细公开的信息的教导下，可以根据例如但不限于上述的具体情况来确定在具体情况下所适用的具体剂量，而无需要作具体限定。特别是，可以参考本发明实施例部分中所用的具体量来确定任一情况下的使用量，例如在本发明下文使用了具体剂量的五指山小型猪PDLSCs，本领域技术人员可根据上述剂量并结合本领域公知技术教导将该剂量换算为人用条件下的剂量。 The actual dose level of tooth-related stem cells in the composition or pharmaceutical composition of the present invention can be changed, so that the amount of stem cells obtained can effectively target specific hosts, patients, and obtain the required dosage under the conditions of specific compositions and their compositions and corresponding administration methods. Treat or prevent reactions. Dosage levels will depend on the activity of the particular stem cells, the route of administration, the severity of the condition being treated, the course of treatment of the disease or condition, the course of formation and repair (as well as generation, regeneration, culture, etc.) Conditions and past medical history were selected. However, it is the practice in the art to start the dosage and timing of stem cells below that required to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained. Therefore, as far as the present invention is concerned, under the teaching of the information disclosed in detail in the present invention, those skilled in the art can determine the specific dosage applicable in a specific case according to, for example but not limited to, the specific conditions described above, without the need for specific limited. In particular, the amount used in any case can be determined with reference to the specific amount used in the Examples of the present invention. For example, the specific dose of Wuzhishan minipig PDLSCs is used below in the present invention. Those skilled in the art can combine the above doses with Known techniques in the art teach converting such doses to those under human conditions. the
本发明牙齿相关干细胞可以单独(即以原样形式)或以药物组合物的形式给药。本发明药物组合物可根据给药途径配成各种适宜的剂型。使用 一种或多种生理学上可接受的载体，包含赋形剂和助剂，它们有利于将牙齿相关干细胞加工成可以在药学上使用的制剂。适当的制剂形式取决于所选择的给药途径，可以按照本领域熟知的常识进行制造。在本发明的一个实施方案中，所述牙齿相关干细胞存在于细胞相容的介质(例如，生理盐水如0.9%生理盐水，等等)中。在本发明的一个实施方案中，所述牙齿相关干细胞存在于细胞相容的介质中，并且在低温下保存，例如在冷藏、冷冻等条件下保存，并且可任选在在临用前复溶成适用根据本发明精神而施用的形式。 The tooth-related stem cells of the present invention can be administered alone (ie, as they are) or in the form of a pharmaceutical composition. The pharmaceutical composition of the present invention can be formulated into various suitable dosage forms according to the route of administration. Using one or more physiologically acceptable carriers, including excipients and auxiliary agents, they facilitate the processing of tooth-related stem cells into pharmaceutically usable preparations. Appropriate formulations depend on the chosen route of administration and can be manufactured according to common knowledge in the art. In one embodiment of the present invention, the tooth-related stem cells are present in a cytocompatible medium (eg, physiological saline such as 0.9% physiological saline, etc.). In one embodiment of the present invention, the tooth-related stem cells are present in a cytocompatible medium and stored at low temperature, such as stored under refrigeration, freezing, etc., and can optionally be reconstituted immediately before use into a form suitable for administration in accordance with the spirit of the invention. the
附图说明 Description of drawings
图1.人PDLSCs免疫分子的表达：应用流式细胞术检测fPDLSCs和cPDLSCs相关免疫分子的表达。HLA-I,HLA-ΠDR,CD80和CD86的表达情况如图所示：fPDLSCs(76.2%±5.8%,n=5)和cPDLSCs(78.5%±6.4%,n=5)表达HLA-I，但是不表达HLA-ΠDR和共刺激分子CD80、CD86。 Figure 1. Expression of immune molecules in human PDLSCs: the expression of immune molecules in fPDLSCs and cPDLSCs was detected by flow cytometry. The expression of HLA-I, HLA-ΠDR, CD80 and CD86 is shown in the figure: fPDLSCs (76.2%±5.8%, n=5) and cPDLSCs (78.5%±6.4%, n=5) express HLA-I, but HLA-ΠDR and co-stimulatory molecules CD80, CD86 are not expressed. the
图2.PDLSCs抑制T淋巴细胞增殖：(a)fPDLSCs/cPDLSC不会引起同种异体T淋巴细胞增殖。(b)fPDLSCs/cPDLSC剂量依赖性地抑制丝裂原PHA引起的T淋巴细胞增殖。(c)延迟加入fPDLSCs/cPDLSC也能够抑制PHA引起的T淋巴细胞增殖。(d)fPDLSCs/cPDLSC能够抑制双向混合淋巴细胞反应。 Figure 2. PDLSCs inhibit the proliferation of T lymphocytes: (a) fPDLSCs/cPDLSCs do not induce proliferation of allogeneic T lymphocytes. (b) fPDLSCs/cPDLSC dose-dependently inhibited the proliferation of T lymphocytes induced by the mitogen PHA. (c) Delayed addition of fPDLSCs/cPDLSC can also inhibit PHA-induced T lymphocyte proliferation. (d) fPDLSCs/cPDLSC can inhibit bidirectional mixed lymphocyte reaction. the
图3.PDLSCs通过分泌PGE2抑制T淋巴细胞增殖：(a)在Transwell培养实验中，PDLSCs也能抑制T淋巴细胞增殖，提示PDLSCs分泌可溶性因子来发挥免疫抑制作用。(b)在单纯PDLSCs培养上清和MLR上清中都存在TGF-β1，但是两者之间没有显著性差异。(c)PGE2的浓度在MLR中显著性升高。(d)中和实验表明抗TGF-β1抗体没有恢复T淋巴细胞增殖，PGE2抑制剂抵消了PDLSCs的免疫抑制作用,提示PGE2是介导PDLSCs免疫抑制作用的主要因子。(e,f)PDLSCs的免疫抑制作用并没有引起T淋巴细胞的凋亡(e),与单纯用PHA刺激T淋巴细胞后的凋亡率一致(f)。(g)PDLSCs引起的被抑制的T淋巴细胞用PHA或者IL-2再刺激时可以重新恢复增殖。 Figure 3. PDLSCs inhibit T lymphocyte proliferation by secreting PGE2: (a) In Transwell culture experiments, PDLSCs can also inhibit T lymphocyte proliferation, suggesting that PDLSCs secrete soluble factors to play an immunosuppressive role. (b) TGF-β1 was present in both pure PDLSCs culture supernatant and MLR supernatant, but there was no significant difference between the two. (c) The concentration of PGE2 was significantly increased in MLR. (d) Neutralization experiments showed that anti-TGF-β1 antibody did not restore T lymphocyte proliferation, and PGE2 inhibitor counteracted the immunosuppressive effect of PDLSCs, suggesting that PGE2 is the main factor mediating the immunosuppressive effect of PDLSCs. (e,f) The immunosuppressive effect of PDLSCs did not induce the apoptosis of T lymphocytes (e), which was consistent with the apoptosis rate after stimulating T lymphocytes with PHA alone (f). (g) Suppressed T lymphocytes induced by PDLSCs can resume proliferation when restimulated with PHA or IL-2. the
图4.HGF和IL-10的测定：在单纯PDLSCs培养上清和MLR上清中都没有测到HGF和IL-10，说明这两种因子没有参与PDLSCs介导 的免疫抑制作用。 Figure 4. Determination of HGF and IL-10: HGF and IL-10 were not detected in the pure PDLSCs culture supernatant and MLR supernatant, indicating that these two factors were not involved in the immunosuppressive effect mediated by PDLSCs. the
图5.PDLSCs介导的牙周组织再生：(a)在-4w和0w,四组的临床指标之间没有显著性的差异。但是治疗后12周,自体或者同种异体PDLSCs移植组的PD,GR和AL与空白对照组和HA/TCP相比显著恢复。(b,c,d,e)CT扫描显示治疗前都有明显的牙周骨缺损，而且缺损程度相近。治疗后12周，自体PDLSCs组(h)和同种异体PDLSCs组(i)完全获得了牙周骨再生。空白对照组几乎没有骨组织再生(f),而HA/TCP组的骨缺损程度加重(g).(j,k,l,m)组织学研究发现自体或者同种异体PDLSCs组在骨缺损区有明显的新骨和牙周组织再生。但是,典型的牙周炎表现如深牙周袋、缺乏新生骨和牙周纤维在HA/TCP组和空白对照组仍然清晰可见。PD:探诊深度,GR:牙龈退缩,AL:附着丧失,D:牙本质,C:牙骨质,PDL:牙周膜,B:骨。 Figure 5. Periodontal tissue regeneration mediated by PDLSCs: (a) At -4w and 0w, there was no significant difference in the clinical indicators of the four groups. However, after 12 weeks of treatment, the PD, GR and AL in the autologous or allogeneic PDLSCs transplantation group were significantly restored compared with the blank control group and HA/TCP. (b,c,d,e) CT scans showed obvious periodontal bone defects before treatment, and the degree of defect was similar. At 12 weeks after treatment, the autologous PDLSCs group (h) and the allogeneic PDLSCs group (i) completely achieved periodontal bone regeneration. There was almost no bone tissue regeneration in the blank control group (f), while the degree of bone defect in the HA/TCP group was aggravated (g). (j, k, l, m) Histological studies found that the autologous or allogeneic PDLSCs group had a greater degree of bone defect in the bone defect area There was significant regeneration of new bone and periodontal tissue. However, typical periodontitis manifestations such as deep periodontal pockets, lack of new bone and periodontal fibers were still clearly visible in the HA/TCP group and the blank control group. PD: probing depth, GR: gingival recession, AL: attachment loss, D: dentin, C: cementum, PDL: periodontal ligament, B: bone. the
图6.同种异体PDLSCs移植不会引起免疫排斥反应：(a)在如图所示的各个时间点，同种异体PDLSCs移植组的CD3+,CD4+,CD8+T淋巴细胞没有显著性差异；(b)在移植后3天，CD3+,CD4+,CD8+T淋巴细胞数目以及活化T淋巴细胞的标志物——CD40L的表达情况在四个治疗组之间没有明显差异。 Figure 6. Allogeneic PDLSCs transplantation will not cause immune rejection: (a) At each time point shown in the figure, there is no significant difference in CD3 + , CD4 + , CD8 + T lymphocytes in the allogeneic PDLSCs transplantation group (b) At 3 days after transplantation, the number of CD3 + , CD4 + , CD8 + T lymphocytes and the expression of CD40L, a marker of activated T lymphocytes, were not significantly different among the four treatment groups.
图7.移植后12w的免疫状况：在治疗后12w，CD3+,CD4+,CD8+T淋巴细胞数目以及活化T淋巴细胞的标志物——CD40L的表达情况在四个治疗组之间也没有明显差异。 Figure 7. Immune status at 12w after transplantation: at 12w after treatment, the number of CD3 + , CD4 + , CD8 + T lymphocytes and the marker of activated T lymphocytes—the expression of CD40L were not different among the four treatment groups Significant differences.
图8.描绘了本发明的一个实施例中PDLSCs介导的牙周组织再生。 Figure 8. Depicts the regeneration of periodontal tissue mediated by PDLSCs in one embodiment of the present invention. the
图9.描绘了在小型猪上成功再生生物牙根。 Figure 9. Depicts successful regeneration of biological tooth roots on minipigs. the
图10.描绘了在小型猪上进行生物牙根再生获得成功。 Figure 10. Depicts the success of biological root regeneration in minipigs. the
图11.描绘了脱落乳牙牙髓干细胞治疗系统性红斑狼疮鼠血清及肾脏组织学改变。 Fig. 11 depicts the histological changes in serum and kidney of rats with systemic lupus erythematosus treated with dental pulp stem cells from deciduous teeth. the
图12.说明同种异体人牙周膜干细胞(hPDLSCs)用于治疗牙周病的标准操作的一个实例。 Figure 12. An example of a standard procedure illustrating the use of allogeneic human periodontal ligament stem cells (hPDLSCs) for the treatment of periodontal disease. the
具体实施方式 Detailed ways
通过下面的实施例可以对本发明进行进一步的描述，然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。 The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. the
本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。 The present invention provides general and/or specific descriptions of the materials and test methods used in the tests. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here in as much detail as possible. the
实施例1：PDLSCs对T淋巴细胞增殖的抑制作用Example 1: Inhibitory effect of PDLSCs on T lymphocyte proliferation
材料和方法Materials and methods
1)、人PDLSCs1), human PDLSCs
选取首都医科大学附属北京口腔医院口腔颌面外科门诊拔除的18-28岁的健康患者的正常第三磨牙，按照以往文献报道的方法分离和培养PDLSCs(3)。人体组织的利用得到首都医科大学伦理委员会的批准。为了下一步实验中应用冻存牙周膜干细胞(cryopreserved periodontal ligament stem cells，cPDLSCs)，将部分新鲜分离的牙周膜干细胞(freshly isolated periodontal ligament stem cells，fPDLSCs)冻存在液氮中3个月。3个月后，冻存细胞在37℃水浴中快速解冻、接种、常规培养。在本研究中，应用的细胞都在第2-4代。在同一实验中，应用同一代的fPDLSCs和cPDLSCs。 The normal third molars of 18-28-year-old healthy patients extracted from the Department of Oral and Maxillofacial Surgery, Beijing Stomatological Hospital Affiliated to Capital Medical University were selected, and PDLSCs were isolated and cultured according to the methods reported in the previous literature (3). The use of human tissue was approved by the Ethics Committee of Capital Medical University. In order to apply cryopreserved periodontal ligament stem cells (cPDLSCs) in the next experiment, some freshly isolated periodontal ligament stem cells (fPDLSCs) were frozen in liquid nitrogen for 3 months. After 3 months, the frozen cells were quickly thawed in a 37°C water bath, inoculated, and routinely cultured. In this study, the cells used were all at passage 2-4. In the same experiment, fPDLSCs and cPDLSCs of the same generation were used. the
2)、外周血单个核细胞2), peripheral blood mononuclear cells
应用梯度密度离心法分离健康供者的外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)。 Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by gradient density centrifugation. the
3)、抗体3), Antibody
本研究中应用抗人白细胞抗原(human leukocyte antigen，HLA)-I、HLA-ΠDR、CD80、CD86抗体(BD Biosciences)，抗CD3、CD4、CD8、CD40L、转化生长因子β(transforming growth factorβ，TGF-β)、肝细胞生长因子(hepatocyte growth factor，HGF)抗体(Abcam),抗IL-10、FITC标记的抗小鼠IgG抗体(AbD Serotec)。 Anti-human leukocyte antigen (HLA)-I, HLA-ΠDR, CD80, CD86 antibodies (BD Biosciences), anti-CD3, CD4, CD8, CD40L, transforming growth factor β (transforming growth factor β, TGF) were used in this study. -β), hepatocyte growth factor (hepatocyte growth factor, HGF) antibody (Abcam), anti-IL-10, FITC-labeled anti-mouse IgG antibody (AbD Serotec).
4)、流式细胞术4), flow cytometry
为了研究PDLSCs表面相关免疫分子的表达，将1.0×106fPDLSCs或者cPDLSCs分别与抗HLA-I、HLA-ΠDR、CD80或者CD86抗体室温下孵育1小时。经过磷酸盐缓冲液(phosphate buffered saline，PBS)冲洗后，细胞再与FITC标记的抗小鼠IgG二抗室温下孵育30分钟。孵育结束后上流式细胞仪(BD Inmmunocytometry Systems)检测表达情况。 In order to study the expression of immune molecules on the surface of PDLSCs, 1.0×10 6 fPDLSCs or cPDLSCs were incubated with anti-HLA-I, HLA-ΠDR, CD80 or CD86 antibodies for 1 hour at room temperature. After washing with phosphate buffered saline (PBS), the cells were incubated with FITC-labeled anti-mouse IgG secondary antibody for 30 minutes at room temperature. After incubation, the expression was detected by flow cytometry (BD Inmmunocytometry Systems).
5)、混合淋巴细胞反应5), mixed lymphocyte reaction
作为刺激细胞，5.0×104fPDLSCs或者cPDLSCs先在直线加速器下照射20Gy，然后加入等细胞量的同种异体PBMCs，在96孔板、0.2mlRPMI-1640中共同孵育5天。在结束反应前18个小时，每孔加入1μCi的3H-thymidine(3H-TdR)。18个小时后，在玻璃纤维滤纸上收集细胞，应用液体闪烁仪(PerkinElmer)计算3H-TdR掺入率。3H-TdR掺入率的结果以每分钟计数±标准差(CPM±SD)来表示。 As stimulator cells, 5.0×10 4 fPDLSCs or cPDLSCs were first irradiated with 20Gy under the linear accelerator, and then the same amount of allogeneic PBMCs was added, and co-incubated in 96-well plates and 0.2ml RPMI-1640 for 5 days. Eighteen hours before the end of the reaction, 1 μCi of 3 H-thymidine ( 3 H-TdR) was added to each well. After 18 hours, the cells were collected on glass fiber filter paper, and the 3 H-TdR incorporation rate was calculated using a liquid scintillation instrument (PerkinElmer). The results of 3 H-TdR incorporation were expressed in counts per minute ± standard deviation (CPM ± SD).
为了研究PDLSCs对增殖T淋巴细胞的影响以及此种影响是否具有剂量依赖性，在丝裂原促增殖实验中，以终浓度为0.5μg/mL的PHA(Sigma-Aldrich)刺激PBMCs(5.0×104)增殖，与不同剂量的自体fPDLSCs或者cPDLSCs共孵育5天(PDLSCs的量分别为1.0×104,5.0×104,2.5×105,5.0×105)。结束孵育前18小时每孔加入1μCi的3H-TdR，18小时后收集细胞并且计算3H-TdR掺入率。 In order to study the effect of PDLSCs on proliferating T lymphocytes and whether this effect is dose-dependent, in the mitogen-promoting experiment, PBMCs (5.0×10 4 ) Proliferate and co-incubate with different doses of autologous fPDLSCs or cPDLSCs for 5 days (the amounts of PDLSCs are 1.0×10 4 , 5.0×10 4 , 2.5×10 5 , and 5.0×10 5 ). Eighteen hours before the end of the incubation, 1 μCi of 3 H-TdR was added to each well, and the cells were collected 18 hours later to calculate the incorporation rate of 3 H-TdR.
进一步研究延迟加入PDLSCs是否会影响T淋巴细胞增殖。先用终浓度为0.5μg/mL的PHA刺激PBMCs(5.0×104)增殖2天，然后加入同等剂量的fPDLSCs或者cPDLSCs再共同孵育3天。3天后测定3H-TdR掺入率。 It was further investigated whether delayed addition of PDLSCs would affect T lymphocyte proliferation. PBMCs (5.0×10 4 ) were first stimulated to proliferate with PHA at a final concentration of 0.5 μg/mL for 2 days, and then the same dose of fPDLSCs or cPDLSCs were added and co-incubated for 3 days. The 3 H-TdR incorporation rate was measured after 3 days.
在证实PDLSCs能够抑制丝裂原PHA引起的T淋巴细胞增殖后，我们观察PDLSCs对双向混合淋巴细胞反应(mixed lymphocyte reaction，MLR)的影响。来自两个不同个体的PBMCs(5.0×104)与等量的第三方fPDLSCs或者cPDLSCs共同孵育5天。结束孵育前18小时每孔加入1μCi的3H-TdR，孵育结束后收集细胞、计算3H-TdR掺入率。 After confirming that PDLSCs can inhibit the proliferation of T lymphocytes induced by mitogen PHA, we observed the effect of PDLSCs on two-way mixed lymphocyte reaction (mixed lymphocyte reaction, MLR). PBMCs (5.0×10 4 ) from two different individuals were co-incubated with an equal amount of third-party fPDLSCs or cPDLSCs for 5 days. Eighteen hours before the end of the incubation, 1 μCi of 3 H-TdR was added to each well, and the cells were collected after the incubation, and the incorporation rate of 3 H-TdR was calculated.
6)、与PDLSCs共孵育后的T淋巴细胞的再次刺激6), re-stimulation of T lymphocytes after co-incubation with PDLSCs
为了观察PDLSCs对T淋巴细胞的增殖抑制是否可逆，我们进行了再激活实验。PBMCs(5.0×104)先与等量的PDLSCs和PHA(0.5μg/mL)共同孵育5天，然后把通过梯度密度离心法获得的T淋巴细胞与PHA(0.5μg/mL)或者重组人白介素2(interleukin2,IL-2,50U/mL;R&D systems)共同反应2天。2天后，应用3H-TdR掺入法测定T淋巴细胞增殖率。 In order to observe whether the inhibition of T lymphocyte proliferation by PDLSCs is reversible, we performed a reactivation experiment. PBMCs (5.0×10 4 ) were first incubated with the same amount of PDLSCs and PHA (0.5 μg/mL) for 5 days, and then the T lymphocytes obtained by gradient density centrifugation were mixed with PHA (0.5 μg/mL) or recombinant human interleukin 2 (interleukin2, IL-2, 50U/mL; R&D systems) reacted together for 2 days. After 2 days, the 3 H-TdR incorporation method was used to measure the proliferation rate of T lymphocytes.
7)、Transwell培养实验7), Transwell culture experiment
为了观察通过细胞-细胞直接接触还是PDLSCs分泌可溶性因子来 介导PDLSCs对T淋巴细胞的增殖抑制，我们进行了Transwell培养实验。Transwell培养系统(Costar)具有直径为0.4μm孔径的微膜，此微膜可以把两种细胞人为地分隔开来。将PBMCs(5.0×104)与PHA(0.5μg/mL)置于Transwell培养系统的上腔,而5.0×104PDLSCs(从本实验开始，本研究的以下部分都用fPDLSCs)接种在下腔。5天后，应用 3H-TdR掺入法测定T淋巴细胞增殖率。 In order to observe whether the inhibition of T lymphocyte proliferation by PDLSCs is mediated by direct cell-cell contact or by secreting soluble factors from PDLSCs, we performed Transwell culture experiments. The Transwell culture system (Costar) has a micro-membrane with a diameter of 0.4 μm, which can artificially separate two kinds of cells. PBMCs (5.0×10 4 ) and PHA (0.5 μg/mL) were placed in the upper chamber of the Transwell culture system, while 5.0×10 4 PDLSCs (fPDLSCs were used in the following parts of this study since the beginning of this experiment) were seeded in the lower chamber of the Transwell culture system. After 5 days, the 3 H-TdR incorporation method was used to measure the proliferation rate of T lymphocytes.
8)、可溶性因子的测定及中和实验8) Determination of soluble factors and neutralization experiment
当发现可溶性因子介导PDLSCs对T淋巴细胞的增殖抑制后，我们应用酶联免疫吸附实验(enzyme-linked immumosorbent assay，ELISA)来测定单纯PDLSCs的培养上清(接种1-5天)和MLR培养上清(5.0×104PDLSCs,等量PBMCs和0.5μg/mL的PHA)中TGF-β1(R&D systems),HGF(R&D systems),前列腺素E2(prostaglandin E2，PGE2;Assay Designs)和IL-10(R&D systems)的浓度。通过酶标仪(Molecules Devices)在450nm(TGF-β1,HGF和IL-10)或者405nm(PGE2)读取光密度值。 When soluble factors were found to mediate the proliferation inhibition of T lymphocytes by PDLSCs, we applied enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) to determine the culture supernatant of simple PDLSCs (inoculation 1-5 days) and MLR culture TGF-β1 ( R &D systems), HGF (R&D systems), prostaglandin E2 (prostaglandin E2, PGE2; Assay Designs) and IL- Concentration of 10 (R&D systems). Optical density values were read at 450 nm (TGF-β1, HGF and IL-10) or 405 nm (PGE2) by a microplate reader (Molecules Devices).
然后进行中和实验，在Transwell培养系统中建立MLR的反应体系，包括PBMCs(5.0×104),PDLSCs(5.0×104)和PHA(0.5μg/mL)，在反应的同时分别加入以下抗体或者试剂：抗TGF-β抗体(10ng/mL),抗HGF抗体(10ng/mL)和抗IL-10抗体(10ng/mL)或者PGE2的抑制剂吲哚美辛(5μmol/L;Sigma-Aldrich)。5天后，通过3H-TdR掺入法测定T淋巴细胞增殖率。 Then carry out the neutralization experiment, establish the MLR reaction system in the Transwell culture system, including PBMCs (5.0×10 4 ), PDLSCs (5.0×10 4 ) and PHA (0.5μg/mL), and add the following antibodies respectively during the reaction Or reagents: anti-TGF-β antibody (10ng/mL), anti-HGF antibody (10ng/mL) and anti-IL-10 antibody (10ng/mL) or PGE2 inhibitor indomethacin (5μmol/L; Sigma-Aldrich ). After 5 days, the proliferation rate of T lymphocytes was measured by 3 H-TdR incorporation method.
9)、测定凋亡T淋巴细胞的百分率9) Determination of the percentage of apoptotic T lymphocytes
建立MLR的反应体系，包括PBMCs(5.0×104),PDLSCs(5.0×104)和PHA(0.5μg/mL)，5天后应用Annexin V-Fluos凋亡试剂盒(Roche Diagnostics)来测定凋亡T淋巴细胞的百分率。 Establish the MLR reaction system, including PBMCs (5.0×10 4 ), PDLSCs (5.0×10 4 ) and PHA (0.5 μg/mL), and use Annexin V-Fluos apoptosis kit (Roche Diagnostics) to measure apoptosis after 5 days Percentage of T lymphocytes.
实施例2：PDLSCs介导的牙周骨缺损修复Example 2: PDLSCs-mediated periodontal bone defect repair
1)、材料和方法1), materials and methods
五指山小型猪和贵州香猪(6-8月龄，体重30-40千克)由中国农业大学实验动物中心提供。本实验通过首都医科大学伦理委员会批准。小型猪尖牙PDLSCs的分离和培养方法同人PDLSCs。按照文献报道的方法(13)，制备12只雌性五指山小型猪的牙周炎骨缺损模型，总共制备24处下颌第一恒磨牙的牙周炎骨缺损。24处缺损随机分为以下4个组：【1】 空白对照组，不做任何治疗；【2】单纯材料组，翻瓣、刮治、移植HA/TCP支架材料(武汉理工大学提供)、明胶海绵覆盖缺损、缝合；【3】自体PDLSCs移植组，翻瓣、刮治、移植HA/TCP支架材料+2.0×107五指山小型猪PDLSCs、明胶海绵覆盖缺损、缝合；【4】异体PDLSCs移植组，翻瓣、刮治、移植HA/TCP支架材料+2.0×107香猪PDLSCs、明胶海绵覆盖缺损、缝合。在制备牙周炎骨缺损模型前(-4w)、移植治疗前(0w)和治疗后12w进行临床检查，包括探诊深度(probing depth，PD)、牙龈退缩(gingival recession，GR)和附着丧失(attachment loss，AL)。在0w和治疗后12w应用CT(Siemens)观察骨再生的情况。在-4w、治疗后1-7d、2w、4w、8w和12w进行血常规检查、血生化检查、免疫球蛋白检查和T淋巴细胞相关标志物的检查，包括CD3+细胞计数、CD4+细胞计数、CD8+细胞计数以及活化T淋巴细胞的标志物——CD40L的表达情况。在治疗后12w，处死动物，从实验部位获取标本，固定、脱钙、石蜡包埋，行HE染色，观察组织再生情况。 Wuzhishan miniature pigs and Guizhou fragrant pigs (6-8 months old, weighing 30-40 kg) were provided by the Experimental Animal Center of China Agricultural University. This experiment was approved by the Ethics Committee of Capital Medical University. The isolation and culture methods of minipig canine PDLSCs are the same as human PDLSCs. According to the method reported in the literature (13), 12 female Wuzhishan minipigs with periodontitis bone defect models were prepared, and a total of 24 mandibular first permanent molars with periodontitis bone defects were prepared. The 24 defects were randomly divided into the following 4 groups: [1] Blank control group, without any treatment; [2] Simple material group, flap flap, curettage, transplantation of HA/TCP scaffold material (provided by Wuhan University of Technology), gelatin Sponge covered defect, sutured; [3] Autologous PDLSCs transplantation group, flap flap, curettage, transplanted HA/TCP scaffold material + 2.0×10 7 Wuzhishan minipig PDLSCs, gelatin sponge covered defect, sutured; [4] Allogeneic PDLSCs transplanted group , Flap flap, curettage, transplantation of HA/TCP scaffold material + 2.0×10 7 Xiangpig PDLSCs, gelatin sponge to cover the defect, and suture. Clinical examinations were performed before the preparation of periodontitis bone defect model (-4w), before transplantation treatment (0w) and 12w after treatment, including probing depth (PD), gingival recession (gingival recession, GR) and attachment loss (attachment loss, AL). CT (Siemens) was used to observe bone regeneration at 0w and 12w after treatment. At -4w, 1-7d, 2w, 4w, 8w and 12w after treatment, blood routine examination, blood biochemical examination, immunoglobulin examination and T lymphocyte related markers examination, including CD3 + cell count, CD4 + cell count , CD8 + cell count and the expression of CD40L, a marker of activated T lymphocytes. 12w after treatment, the animals were sacrificed, and specimens were obtained from the experimental site, fixed, decalcified, embedded in paraffin, and stained with HE to observe the tissue regeneration.
通过Student’t检验和方差分析进行统计学分析,p<0.05认为是有统计学差异。 Statistical analysis was carried out by Student't test and analysis of variance, and p<0.05 was considered to be statistically different. the
2)、结果和说明2), results and description
2-1)、PDLSCs具有低免疫原性2-1), PDLSCs have low immunogenicity
首先观察到人PDLSCs的免疫表型，发现fPDLSCs(76.2%±5.8%,n=5)和cPDLSCs(78.5%±6.4%,n=5)表达HLA-I，但是不表达HLA-ΠDR和共刺激分子CD80、CD86(图1)，与体外培养扩增的BMSCs表达状况相似(23)。 The immunophenotype of human PDLSCs was first observed, and fPDLSCs (76.2%±5.8%, n=5) and cPDLSCs (78.5%±6.4%, n=5) were found to express HLA-I, but not HLA-ΠDR and co-stimulatory Molecules CD80 and CD86 (Figure 1) are similar to those expressed in BMSCs cultured and expanded in vitro (23). the
进一步研究PDLSCs作为抗原提呈细胞对T淋巴细胞增殖的影响。实验组为预先经过直线加速器20Gy照射的5.0×104PDLSCs与等量的同种异体PBMCs共孵育。作为阳性对照的T淋巴细胞增殖组为：5.0×104PBMCs与与等量的同种异体PBMCs共孵育。单独培养等量的PBMCs为阴性对照。结果表明，实验组PDLSCs没有引起同种异体PBMCs增殖，而阳性对照组可以引起显著的 To further study the effect of PDLSCs as antigen-presenting cells on the proliferation of T lymphocytes. The experimental group was co-incubated with 5.0×10 4 PDLSCs pre-irradiated by a linear accelerator at 20Gy and the same amount of allogeneic PBMCs. The T lymphocyte proliferation group as a positive control: 5.0×10 4 PBMCs were co-incubated with the same amount of allogeneic PBMCs. The same amount of PBMCs cultured alone served as negative control. The results showed that PDLSCs in the experimental group did not cause the proliferation of allogeneic PBMCs, while the positive control group could cause significant
T淋巴细胞增殖(图2a)，提示PDLSCs具有低免疫原性。 T lymphocytes proliferated (Fig. 2a), suggesting that PDLSCs have low immunogenicity. the
2-2)、PDLSCs能够抑制T淋巴细胞增殖2-2), PDLSCs can inhibit the proliferation of T lymphocytes
进一步观察PDLSCs对丝裂原和同种异体抗原引起的T淋巴细胞增殖的影响。将fPDLSCs和cPDLSCs接种，细胞量分别为：1.0×104, 5.0×104,2.5×105和5.0×105，然后加入自体PBMCs(5.0×104)和终浓度为0.5μg/mL的PHA。PHA刺激PBMCs(5.0×104)增殖为阳性对照。结果发现，PHA刺激的PBMCs增殖明显地被fPDLSCs或者cPDLSCs所抑制，而且这种抑制是剂量依赖性的。此种免疫抑制作用必须要求PDLSCs的存在，而不是由体积效应(bulk effect)引起，因为在增殖反应体系中加入等量的自体PBMCs不会引起抑制作用(图2b)。此外，即使在PHA刺激PBMCs(5.0×104)增殖后两天再加入PDLSCs，仍然能够明显抑制T淋巴细胞的增殖(图2c)。我们进一步观察PDLSCs对MLR的影响。来自两个不同个体的PBMCs和第三方的PDLSCs共同孵育。结果表明，fPDLSCs和cPDLSCs都能够抑制双向MLR(图2d)。本部分实验数据表明，PDLSCs能够剂量依赖性地和抗原非特异性地抑制同种异体T细胞受体引起的和丝裂原引起的T淋巴细胞增殖。 Further observe the effects of PDLSCs on the proliferation of T lymphocytes induced by mitogens and alloantigens. The fPDLSCs and cPDLSCs were inoculated with cell volumes of 1.0×10 4 , 5.0×10 4 , 2.5×10 5 and 5.0×10 5 , and then autologous PBMCs (5.0×10 4 ) and a final concentration of 0.5 μg/mL of PHAs. PHA stimulated the proliferation of PBMCs (5.0×10 4 ) as a positive control. It was found that PHA-stimulated PBMCs proliferation was significantly inhibited by fPDLSCs or cPDLSCs, and this inhibition was dose-dependent. This immunosuppressive effect must require the presence of PDLSCs, rather than caused by the bulk effect (bulk effect), because adding the same amount of autologous PBMCs to the proliferation response system did not cause the inhibitory effect (Fig. 2b). In addition, even adding PDLSCs two days after PHA stimulated the proliferation of PBMCs (5.0×10 4 ) could still significantly inhibit the proliferation of T lymphocytes ( FIG. 2 c ). We further observed the effect of PDLSCs on MLR. PBMCs from two different individuals were co-incubated with PDLSCs from a third party. The results showed that both fPDLSCs and cPDLSCs were able to inhibit bidirectional MLR (Fig. 2d). The experimental data in this part show that PDLSCs can dose-dependently and antigen-nonspecifically inhibit allogeneic T cell receptor-induced and mitogen-induced T lymphocyte proliferation.
2-3)、PDLSCs通过分泌PGE2抑制T淋巴细胞增殖2-3), PDLSCs inhibit T lymphocyte proliferation by secreting PGE2
我们进一步研究PDLSCs抑制T淋巴细胞增殖的机制问题。为了阐明是否通过PDLSCs和PBMCs细胞-细胞直接接触引起，我们首先进行了Transwell培养实验，将PDLSCs和PBMCs人为地分隔开来。研究结果表明，无论是Transwell培养实验还是细胞-细胞直接接触实验，都能够取得相近的T淋巴细胞增殖抑制效果，提示这种免疫抑制作用与细胞-细胞直接接触无关，而是依靠可溶性因子的存在(图3a)。 We further studied the mechanism of PDLSCs inhibiting the proliferation of T lymphocytes. To elucidate whether it is caused by direct cell-cell contact between PDLSCs and PBMCs, we first performed Transwell culture experiments to artificially separate PDLSCs and PBMCs. The results of the study showed that both the Transwell culture experiment and the direct cell-cell contact experiment could achieve similar inhibitory effects on the proliferation of T lymphocytes, suggesting that this immunosuppressive effect has nothing to do with direct cell-cell contact, but relies on the presence of soluble factors (Fig. 3a). the
通过ELISA，我们测定单纯PDLSCs培养上清中和MLR上清中几种可能性的可溶性因子。无论PDLSCs培养上清还是MLR上清，都没有发现HGF和IL-10(图4)。然后我们继续观察TGF-β1和PGE2，因为这两者被认为是BMSCs发挥免疫调节功能的主要因子(7,25)。如图3b和图3c所示,在接种后1-5d的PDLSCs培养上清中，TGF-β1和PGE2的浓度相对稳定，没有明显的波动。但是，在MLR上清中，PGE2的浓度达15.19±1.26ng/ml，与单纯PDLSCs培养的浓度相比明显升高，TGF-β1的浓度为1459.79±109.49pg/mL，没有明显变化。 By ELISA, we assayed several possible soluble factors in culture supernatants of pure PDLSCs and in MLR supernatants. Neither HGF nor IL-10 was found in the culture supernatant of PDLSCs or MLR (Fig. 4). We then went on to look at TGF-β1 and PGE2, since these two are considered to be the main factors for BMSCs to exert immunomodulatory functions (7,25). As shown in Figure 3b and Figure 3c, in the PDLSCs culture supernatant 1-5d after inoculation, the concentrations of TGF-β1 and PGE2 were relatively stable without obvious fluctuations. However, in the supernatant of MLR, the concentration of PGE2 reached 15.19±1.26ng/ml, which was significantly higher than that of pure PDLSCs cultured, and the concentration of TGF-β1 was 1459.79±109.49pg/mL, which did not change significantly. the
我们通过在MLR体系中添加特异性的抗TGF-β,IL-10,HGF抗体和PGE2抑制剂来观察其恢复T淋巴细胞增殖的能力，进一步确定PDLSCs抑制T淋巴细胞增殖的主要因子。结果发现，加入PGE2抑制剂能够显著恢复T淋巴细胞的增殖，而且此时的增殖力同单纯用PHA刺激PBMCs的增殖力相近(图3d)。而实验体系中加入抗TGF-β,IL-10, HGF的中和抗体并没有恢复T淋巴细胞的增殖(图3d)。这些数据表明PGE2是介导PDLSCs抑制T淋巴细胞增殖的主要因子。 We added specific anti-TGF-β, IL-10, HGF antibodies and PGE2 inhibitors to the MLR system to observe its ability to restore T lymphocyte proliferation, and further determined the main factors that PDLSCs inhibited T lymphocyte proliferation. It was found that the addition of PGE2 inhibitors could significantly restore the proliferation of T lymphocytes, and the proliferation ability at this time was similar to that of PBMCs stimulated by PHA alone (Fig. 3d). However, the addition of neutralizing antibodies against TGF-β, IL-10, and HGF in the experimental system did not restore the proliferation of T lymphocytes (Fig. 3d). These data suggest that PGE2 is the main factor mediating the inhibition of T lymphocyte proliferation by PDLSCs. the
就BMSCs的免疫抑制机制而言，虽然目前尚不清楚哪一种因子发挥主导性的作用以及某些研究还存在不一致，但是大多数的研究认识还是通过BMSCs分泌抗增殖的可溶性因子比如IL-10,HGF,TGF-β1,PGE2,吲哚胺-2,3-二氧化酶和一氧化氮(5,6,17,19,20,24-28)引起。Beyth等(28)报道IL-10是BMSCs免疫抑制作用的主要介导因子。DiNicola等(7)通过抗体中和实验发现TGF-β1和HGF是发挥作用的主要因素。另有研究表明PGE2的抑制剂能够抵消BMSCs的免疫抑制作用(25)。这些研究都表明了可溶性因子参与了BMSCs的免疫抑制作用。我们的研究提示PGE2是PDLSCs抑制T淋巴细胞增殖的主要因子。 As far as the immunosuppressive mechanism of BMSCs is concerned, although it is not clear which factor plays a leading role and some studies are still inconsistent, most studies still know that BMSCs secrete anti-proliferative soluble factors such as IL-10 , HGF, TGF-β1, PGE2, indoleamine-2,3-dioxygenase and nitric oxide (5,6,17,19,20,24-28). Beyth et al. (28) reported that IL-10 is the main mediator of the immunosuppressive effect of BMSCs. DiNicola et al. (7) found that TGF-β1 and HGF are the main factors that play a role through antibody neutralization experiments. Another study showed that inhibitors of PGE2 could counteract the immunosuppressive effect of BMSCs (25). These studies have shown that soluble factors are involved in the immunosuppressive effect of BMSCs. Our study suggested that PGE2 is the main factor for PDLSCs to inhibit the proliferation of T lymphocytes. the
2-4)、PDLSCs的免疫抑制作用与T细胞凋亡无关，而是诱导T细胞无能2-4), the immunosuppressive effect of PDLSCs has nothing to do with T cell apoptosis, but induces T cell anergy
进一步研究PDLSCs的免疫抑制作用是否引起了T淋巴细胞的凋亡。现发现，在PDLSCs、PBMCs和PHA的反应体系中，凋亡的T淋巴细胞比例与单纯PBMC、PHA反应体系相似，排除了T淋巴细胞凋亡是引起免疫抑制的可能性(图3e,f)。然后，把被PDLSCs抑制了5天的T淋巴细胞提取，用PHA或者IL-2再刺激两天。结果发现，已经被抑制了的T淋巴细胞再次明显增殖，这种增殖程度与单纯PHA刺激PBMCs的程度相近似(图3g)。因此，可以认为PDLSCs的T淋巴细胞增殖抑制是可逆的，通过诱导T淋巴细胞无能引起。 Further study whether the immunosuppressive effect of PDLSCs caused the apoptosis of T lymphocytes. It was found that in the reaction system of PDLSCs, PBMCs and PHA, the proportion of apoptotic T lymphocytes was similar to that of pure PBMC and PHA reaction system, which ruled out the possibility that T lymphocyte apoptosis caused immunosuppression (Fig. 3e, f) . Then, the T lymphocytes suppressed by PDLSCs for 5 days were extracted and stimulated with PHA or IL-2 for another two days. It was found that the suppressed T lymphocytes re-proliferated significantly, and the degree of proliferation was similar to that of PBMCs stimulated by PHA alone (Fig. 3g). Therefore, it can be considered that the inhibition of T lymphocyte proliferation by PDLSCs is reversible, caused by the induction of T lymphocyte anergy. the
2-5)、PDLSCs介导的牙周组织再生2-5), PDLSCs-mediated periodontal tissue regeneration
鉴于PDLSCs的免疫调节特性，研究同种异体PDLSCs能否修复小型猪牙周炎骨缺损模型。在移植后12周，同种异体移植PDLSCs组的PD为3.5±0.6mm,自体移植组的PD为3.3±0.4mm,HA/TCP组为13.1±1.1mm,空白对照组为10.6±1.3mm。统计学分析表明自体或者同种异体PDLSCs移植组与HA/TCP组和空白对照组相比牙周组织得到了明显再生,而自体或者同种异体PDLSCs组之间并没有显著差异(图5a)。CT扫描显示自体和同种异体PDLSCs组的牙槽骨明显再生，基本恢复到了正常水平，而HA/TCP组和空白对照组仅仅少量再生或者没有再生(图5a-h)。组织学观察表明，在自体和同种异体PDLSCs组，明显再生出新骨、牙骨质和牙周纤维，在HA/TCP组和空白对照组，仍 可见明显的牙周炎表现，包括深牙周袋、缺乏新生骨和牙周纤维(图5j-m)。此外，与移植治疗前4w相比，治疗后各时间点的血常规检查(表1)、血生化检查(表2)、免疫球蛋白检查(表3)和免疫学相关指标(图6，图7)都没有明显变化，表明同种异体移植PDLSCs修复牙周炎骨缺损没有排斥反应的发生。 In view of the immunomodulatory properties of PDLSCs, it was investigated whether allogeneic PDLSCs could repair bone defects in a minipig model of periodontitis. At 12 weeks after transplantation, the PD of the allograft PDLSCs group was 3.5±0.6mm, that of the autologous transplantation group was 3.3±0.4mm, that of the HA/TCP group was 13.1±1.1mm, and that of the blank control group was 10.6±1.3mm. Statistical analysis showed that the autologous or allogeneic PDLSCs transplantation group was significantly regenerated compared with the HA/TCP group and the blank control group, but there was no significant difference between the autologous or allogeneic PDLSCs groups (Figure 5a). CT scans showed that the alveolar bone in the autologous and allogeneic PDLSCs groups regenerated significantly, and basically returned to normal levels, while the HA/TCP group and the blank control group had only a small amount of regeneration or no regeneration (Fig. 5a-h). Histological observations showed that in the autologous and allogeneic PDLSCs groups, new bone, cementum, and periodontal fibers were obviously regenerated. Pockets, lack of new bone and periodontal fibers (Fig. 5j-m). In addition, compared with 4 weeks before transplantation treatment, blood routine examination (Table 1), blood biochemical examination (Table 2), immunoglobulin examination (Table 3) and immunological related indicators (Fig. 6, Fig. 7) There was no significant change, indicating that allograft PDLSCs repaired periodontitis bone defects without rejection. the
表1 Table 1
表2 Table 2
表3 table 3
此外，根据本实施例的另一次试验结果见图8，其中详细描绘了PDLSCs介导的牙周组织再生，从图中显示的结果表明，与本实施例中上面的试验结果一致。 In addition, another test result according to this embodiment is shown in Fig. 8, which depicts the periodontal tissue regeneration mediated by PDLSCs in detail, and the results shown in the figure show that it is consistent with the above test results in this embodiment. the
总之，本发明表明，PDLSCs表达HLA-Ⅰ,不表达HLA-ⅡDR、CD80和CD86，也不会引起同种异体T淋巴细胞增殖，提示PDLSCs具有低免疫原性；PDLSCs能够抑制丝裂原和同种异体抗原引起的T淋巴细胞增殖；PDLSCs不会引起T淋巴细胞的凋亡，T淋巴细胞再次受到刺激时可以恢复增殖能力；PDLSCs通过分泌PGE2来发挥其免疫抑制功能；同种异体PDLSCs可以修复小型猪牙周炎骨缺损模型，且不会引起免疫排斥反应。 In a word, the present invention shows that PDLSCs express HLA-I, do not express HLA-II DR, CD80 and CD86, and will not cause proliferation of allogeneic T lymphocytes, suggesting that PDLSCs have low immunogenicity; PDLSCs can inhibit mitogen and syngeneic Proliferation of T lymphocytes caused by a heterogeneous antigen; PDLSCs will not cause apoptosis of T lymphocytes, and T lymphocytes can restore their proliferation ability when stimulated again; PDLSCs exert their immunosuppressive function by secreting PGE2; allogeneic PDLSCs can repair A bone defect model of periodontitis in minipigs without immune rejection. the
实施例3：牙齿相关间充质干细胞介导的生物牙根再生Example 3: Biological root regeneration mediated by tooth-associated mesenchymal stem cells
1)材料和方法1) Materials and methods
1-1)种子细胞的分离、培养1-1) Isolation and cultivation of seed cells
根尖牙乳头干细胞：麻醉下无菌拔除小型猪尖牙，切取根尖部分根尖牙乳头，用D-Hank's液分别反复清洗，剪碎，置于含Ⅰ型胶原酶(3g/L)和Dispase(4g/L)的消化液，37℃下消化1h，过70μm滤网收集细胞，1000r/min离心10min，用培养液重新悬浮成单细胞悬液。按0.01～1×105/孔分别将根尖牙乳头细胞及牙周膜细胞接种于6孔板中，在α-MEM培养基(含15%胎牛血清，2mmol/L谷氨酰胺，100U/ml青霉素，100μg/ml链霉素)37℃、5%CO2培养，每2～3天换液1次。达80％融合时传代培 养。 Stem cells of the apical papilla: under anesthesia, the canines of miniature pigs were aseptically extracted, and the apical part of the apical papilla was excised, washed repeatedly with D-Hank's solution, chopped, placed in a medium containing collagenase type Ⅰ (3g/L) and Dispase (4g/L) digestion solution was digested at 37°C for 1h, the cells were collected through a 70μm filter, centrifuged at 1000r/min for 10min, and resuspended with culture medium to form a single cell suspension. Apical papilla cells and periodontal ligament cells were inoculated in 6-well plates at 0.01～1×10 5 /well, respectively, in α-MEM medium (containing 15% fetal bovine serum, 2mmol/L glutamine, 100U /ml penicillin, 100μg/ml streptomycin) cultured at 37°C, 5% CO 2 , changing the medium once every 2-3 days. Subculture when reaching 80% confluency.
牙髓干细胞：麻醉下无菌拔除小型猪尖牙，劈开牙齿取牙髓组织，用D-Hank's液分别反复清洗，剪碎，消化，培养，具体步骤同根尖牙乳头细胞。 Dental pulp stem cells: Miniature pig canines were aseptically extracted under anesthesia, the teeth were split open to obtain dental pulp tissue, washed with D-Hank's solution repeatedly, chopped, digested, and cultured, the specific steps were the same as those for apical papilla cells.
牙周膜干细胞：麻醉下无菌拔除小型猪尖牙，轻轻剥离其周围的牙周组织，取中段的牙周组织，用D-Hank's液分别反复清洗，剪碎，消化，培养，具体步骤同根尖牙乳头细胞。 Periodontal ligament stem cells: Under anesthesia, the canines of miniature pigs were aseptically extracted, and the surrounding periodontal tissue was gently peeled off, and the middle periodontal tissue was taken, washed repeatedly with D-Hank's solution, chopped, digested, and cultured, the specific steps Same root canine papilla cells.
1-2)牙周膜细胞膜片制备1-2) Preparation of periodontal ligament cell sheets
将生长旺盛的2×105第二或第三代牙周膜干细胞接种在60mm培养皿，培养成分为α-MEM培养基(含15%胎牛血清，100μmol/L的L-抗坏血酸2-磷酸，2mmol/L谷氨酰胺，100U/ml青霉素，100μg/ml链霉素)。培养10—14天，培养皿边缘细胞出现皱褶，用较钝刀片或细胞刮子将细胞膜片整体揭下来，过程中细胞膜片不要干燥。 Inoculate vigorously growing 2×10 5 second or third generation periodontal ligament stem cells on a 60 mm culture dish, and the culture components are α-MEM medium (containing 15% fetal bovine serum, 100 μmol/L L-ascorbic acid 2-phosphate , 2mmol/L glutamine, 100U/ml penicillin, 100μg/ml streptomycin). After culturing for 10-14 days, the cells on the edge of the culture dish are wrinkled. Use a blunt blade or a cell scraper to peel off the cell membrane as a whole, and the cell membrane should not be dried during the process.
1-3)支架材料1-3) Bracket material
将羟基磷灰石/磷酸三钙支架材料做成类似牙根的外形，尺寸为：直径5mm，长度15mm的圆锥体。羟基磷灰石/磷酸三钙为多孔网状结构,孔径多为200～500μm，牙髓干细胞复合在羟基磷灰石/磷酸三钙三维支架上，在生物反应器内生长第5天的牙髓干细胞，细胞充分伸展，细胞表面的突起和分泌颗粒很多，，连接成一片，直径多在20-50μm。 The hydroxyapatite/tricalcium phosphate scaffold material is made into a shape similar to a tooth root, and the size is a cone with a diameter of 5 mm and a length of 15 mm. Hydroxyapatite/tricalcium phosphate is a porous network structure with a pore size of 200-500 μm. Dental pulp stem cells are compounded on the hydroxyapatite/tricalcium phosphate three-dimensional scaffold. Dental pulp grown on the fifth day in a bioreactor For stem cells, the cells are fully stretched, and there are many protrusions and secretory granules on the cell surface, which are connected into a piece, and the diameter is mostly 20-50 μm. the
1-4)回植前细胞复合1-4) Cell compounding before replantation
将1×108培养至第3代的根尖牙乳头干细胞悬于培养基，将牙根型支架材料置于含细胞的培养基，在37°C摇床上充分混合2h，将支架材料小心夹出，放置于培养皿中，将剩余的细胞悬液小心滴加于支架材料上，静置4h后加培养液，在生物反应器内培养5d后回植。 Suspend 1× 108 apical papilla stem cells cultured to passage 3 in the culture medium, place the root scaffold material in the cell-containing medium, mix thoroughly on a shaker at 37°C for 2 hours, and carefully clamp out the scaffold material , placed in a petri dish, carefully drop the remaining cell suspension onto the scaffold material, add the culture solution after standing for 4 hours, and replant after culturing in the bioreactor for 5 days.
1-5)回植方法1-5) Replanting method
分别选取小型猪的缺牙区。切开黏膜，翻开黏骨膜用瓣，暴露牙槽嵴顶。用种植机以800转/分钟的速度在牙槽骨内钻一个与羟基磷灰石/磷酸三钙支架材料形状相似的孔，将上述根尖牙乳头干细胞/牙髓干细胞与牙根形羟基磷灰石/磷酸三钙混合后在生物反应器内培养5—7天，牙周膜干细胞膜片包绕羟基磷灰石/磷酸三钙表面，回植于小型猪牙槽骨内。 The edentulous areas of minipigs were selected respectively. The mucosa was incised, and the mucoperiosteal flap was opened to expose the alveolar crest. Drill a hole in the alveolar bone with a planter at a speed of 800 rpm, which is similar in shape to the hydroxyapatite/tricalcium phosphate scaffold material, and combine the above-mentioned apical papilla stem cells/dental pulp stem cells with the root-shaped hydroxyapatite The stone/tricalcium phosphate mixture was cultured in a bioreactor for 5-7 days, and the periodontal ligament stem cell membrane was wrapped around the surface of hydroxyapatite/tricalcium phosphate, and replanted in the alveolar bone of miniature pigs. the
2）结果和讨论2) Results and Discussion
牙周膜细胞片应用于牙周组织工程已经有一段时间的历史，但常规采 用Recell温度响应培养皿，培养皿的表面使用了一种温度敏感的疏水材料PIPA-Am。当温度高于32℃时，表面具有疏水性，适合细胞的附着和生长，当温度降低，聚合体变得亲水及膨胀，自然释放细胞。收获细胞只需将温度降到20℃即可，无需酶消化和处理，可以保留细胞表面功能和活性。但培养皿需要特殊的材料PIPA-Am，因此我们摸索出了应用普通的培养皿制备牙周膜细胞膜片的方法，培养基成分中加入100μmol/L的L-抗坏血酸2-磷酸，可以促进细胞快速增殖，并能分泌大量细胞外基质胶原成分，将所有细胞连接在一起，生长到一定程度后，我们将整个细胞膜片完整揭下来。细胞膜片不破坏细胞外基质连接形成的支架，不破坏细胞表面蛋白的粘附增殖及分化等功能，可促进回植体内后形成牙周组织。正常牙周膜特别薄约0.2mm，如果采用支架材料，相对较厚，材料降解后遗留的空隙是个问题，牙周膜片避免了此问题。 Periodontal ligament cell sheets have been used in periodontal tissue engineering for some time, but Recell temperature-responsive culture dishes are routinely used, and a temperature-sensitive hydrophobic material PIPA-Am is used on the surface of the culture dish. When the temperature is higher than 32°C, the surface is hydrophobic, which is suitable for cell attachment and growth. When the temperature is lowered, the polymer becomes hydrophilic and expands, releasing cells naturally. Harvesting cells only needs to lower the temperature to 20°C, without enzymatic digestion and treatment, and can retain cell surface functions and activities. However, the petri dish requires a special material PIPA-Am, so we explored a method to prepare periodontal ligament cell membranes using ordinary petri dishes. Adding 100 μmol/L L-ascorbic acid 2-phosphate to the medium components can promote the rapid development of cells. Proliferate, and can secrete a large amount of extracellular matrix collagen components to connect all cells together. After growing to a certain extent, we will completely peel off the entire cell membrane. The cell membrane does not destroy the scaffold formed by the connection of the extracellular matrix, does not destroy the adhesion, proliferation and differentiation of the cell surface proteins, and can promote the formation of periodontal tissue after implantation. The normal periodontal ligament is particularly thin at about 0.2 mm. If the scaffold material is used, it is relatively thick. The gap left by the material degradation is a problem. The periodontal ligament avoids this problem. the
由于重力及营养供应的因素，对细胞支架复合物进行静止性三维培养的过程中，发现细胞多聚集到支架材料的底部和表面，材料内部黏附的细胞数量很少。培养过程中，静止培养还限制了细胞生长的深度，而牙髓干细胞在支架材料内部均匀分布，才能形成均匀一致的组织。想要让细胞能在支架材料内部也能很好的生长，营养物质能够有效的在支架内部传输和及时的排出细胞代谢废物很重要。生物反应器虽然还不能模拟体内循环系统的物质交换机制，但可在支架材料内部产生流体力学环境，如果控制好力学强度的大小，既可减少对细胞的损伤作用，又可为细胞的生长提供有利的力学环境。本实验中细胞复合到材料后在生物反应器内培养5天，生物反应器动力性三维培养促进了支架内部营养物质的交换，促进了种子细胞在支架材料中的均匀分布，有利于细胞保持成骨表型及促进细胞外基质在支架材料中的沉积。 Due to the factors of gravity and nutrient supply, during the static three-dimensional culture of the cell-scaffold complex, it was found that the cells mostly aggregated to the bottom and surface of the scaffold material, and the number of cells adhered inside the material was very small. During the culture process, the static culture also limits the depth of cell growth, and the dental pulp stem cells are evenly distributed inside the scaffold material to form a uniform and consistent tissue. In order for cells to grow well inside the scaffold material, it is very important that nutrients can be effectively transported inside the scaffold and cell metabolic waste can be discharged in a timely manner. Although the bioreactor cannot simulate the material exchange mechanism of the circulatory system in the body, it can generate a hydrodynamic environment inside the scaffold material. If the mechanical strength is well controlled, it can not only reduce the damage to the cells, but also provide a good environment for the growth of the cells. Favorable mechanical environment. In this experiment, the cells were compounded into the material and cultured in the bioreactor for 5 days. The dynamic three-dimensional culture of the bioreactor promoted the exchange of nutrients inside the scaffold, promoted the uniform distribution of seed cells in the scaffold material, and was conducive to the maintenance of cell growth. Bone phenotype and promotion of extracellular matrix deposition in scaffold materials. the
回植6～9月后，回植的生物牙根在X线片表现为不透射的高密度影，周围有低密度影像环绕。组织学上，再生的生物牙根组织形态与新生的骨组织完全不同，是由许多处于不同生长阶段的球形硬组织团块组成，球形团块相互连接成网状。新生的球形硬组织体积较小，外面围绕着疏松排列呈蓝紫色的细胞，推测可能是成牙本质细胞或成牙骨质细胞，中央为均匀、粉染的基质。较成熟的球形硬组织团块体积较大，中间基本不含细胞，或可见极少量被包埋于其中的细胞碎片。团块中有类牙本质样的结构，其中可见排列不规则的牙本质小管样结构，也有类似牙骨质样的结构，镜下表 现为较均匀一致的硬组织。新形成的硬组织区域外为纤维结缔组织包绕，未见炎细胞浸润，部分区域内还可见类似Sharp's纤维样的结构插入正在形成的类牙骨质和牙本质样结构中。 Six to nine months after the replantation, the replanted biological root appeared as an opaque high-density shadow surrounded by low-density images on X-ray films. Histologically, the morphology of the regenerated biological root tissue is completely different from that of newborn bone tissue. It is composed of many spherical hard tissue clumps in different growth stages, and the spheroidal clumps are interconnected to form a network. The newly formed spherical hard tissue is small in size, surrounded by loosely arranged blue-purple cells, which may be odontoblasts or cementoblasts, and a uniform, pink-stained matrix in the center. The more mature spherical hard tissue mass is larger in size, basically free of cells in the center, or a very small amount of cell fragments embedded in it can be seen. There are dentin-like structures in the mass, in which irregularly arranged dentine tubule-like structures can be seen, and there are also cementum-like structures, which appear as more uniform and consistent hard tissues under the microscope. The newly formed hard tissue area was surrounded by fibrous connective tissue, and no inflammatory cell infiltration was seen. In some areas, structures similar to Sharp's fibers were inserted into the cementoid and dentin-like structures that were forming. the
由上述公开的技术方案可见，本发明对于利用生物牙根再生修复牙齿缺失有十分重要和深远的意义：首先，本发明提出了基于组织工程技术的生物牙根再生的新理念。其次，本发明将生物牙根再生的新理念在大型动物小型猪上实施，获得成功，证明该发明方法可行，有望成为一种新的修复方式应用于临床。第三，本发明提供的具体实施方法系统、可靠，为进行生物牙根再生的深入研究及商品化提供理论依据和技术支持。 From the technical solution disclosed above, it can be seen that the present invention has very important and far-reaching significance for the restoration of tooth loss by biological tooth root regeneration: First, the present invention proposes a new concept of biological tooth root regeneration based on tissue engineering technology. Secondly, the present invention implements the new concept of biological tooth root regeneration on large animals such as minipigs, and has achieved success, which proves that the inventive method is feasible and is expected to become a new restoration method for clinical application. Third, the specific implementation method provided by the present invention is systematic and reliable, and provides theoretical basis and technical support for in-depth research and commercialization of biological tooth root regeneration. the
在本实施例方法中，提供的一个实验结果见图9所示，其中详细描绘了在小型猪上成功再生生物牙根的情况。其中，A：实验流程图；（B,C）：形成了牙本质牙骨质样结构及牙周膜样结构（HE染色）；D：牙周膜COLⅠ染色阳性；E：牙本质牙骨质样结构Dsp染色阳性；F～I：再生生物牙根扫描电镜（SEM）观察：F：HA/TCP材料呈多孔隙结构（100-400um）；H：新形成的牙周膜纤维插入新形成的牙本质牙骨质结构及牙槽骨内（G图箭头所指）；I：新形成的牙本质样结构（G图粗箭头所指）。 In the method of this example, the results of an experiment provided are shown in Figure 9, which depicts in detail the successful regeneration of biological tooth roots on minipigs. Among them, A: the experimental flow chart; (B, C): the formation of dentin cementum-like structure and periodontal ligament-like structure (HE staining); D: positive staining of periodontal ligament COLⅠ; E: dentin cementum Dsp-like structure was positively stained; F～I: Scanning electron microscope (SEM) observation of regenerated biological tooth roots: F: HA/TCP material showed a porous structure (100-400um); H: Newly formed periodontal ligament fibers inserted into newly formed teeth Dental cementum structure and alveolar bone (indicated by the arrow in G); I: newly formed dentin-like structure (indicated by the thick arrow in G). the
在本实施例方法中，提供的另一个实验结果见图10所示，与图9类似，图10中详细描绘了在小型猪上进行生物牙根再生获得成功的情况。 In the method of this embodiment, another experimental result provided is shown in FIG. 10 , which is similar to FIG. 9 . FIG. 10 depicts in detail the successful situation of biological tooth root regeneration on minipigs. the
实施例4：脱落乳牙牙髓干细胞移植治疗系统性红斑狼疮(SLE)Example 4: Exfoliated deciduous tooth pulp stem cell transplantation for the treatment of systemic lupus erythematosus (SLE)
1)材料和方法1) Materials and methods
1-1)人SHED1-1) Human SHED
选取拔除的6-8岁的健康患者的正常乳牙，按照以往文献报道的方法分离和培养SHED(3)。人体组织的利用得到首都医科大学伦理委员会的批准。 The extracted normal deciduous teeth of healthy patients aged 6-8 were selected, and SHED was isolated and cultured according to the methods reported in the previous literature (3). The use of human tissue was approved by the Ethics Committee of Capital Medical University. the
1-2)人骨髓基质干细胞和外周血单个核细胞1-2) Human bone marrow stromal stem cells and peripheral blood mononuclear cells
应用梯度密度离心法分离健康供者的骨髓基质干细胞(bone marrow mesenchymal stem cells，BMMSCs)和外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)。 Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by gradient density centrifugation. the
1-3)SHED治疗系统性红斑狼疮(SLE)1-3) SHED for the treatment of systemic lupus erythematosus (SLE)
C57BL/6J and C3MRL-Faslpr/J(MRL/lpr)小鼠(雌性,6-7周龄),Beige nude/nude Xid(III)小鼠(雌性,8-12周龄)由提供。本实验通过首 都医科大学伦理委员会批准。实验分为以下3个组：1、空白对照组(n=3)，不做任何治疗。2、移植BMMSCs组(n=3)。3、移植SHED组(n=3)。在全麻下，将SHED或BMMSCs(1x105cells/10g体重，细胞悬浮于100ml PBS)通过尾静脉注射入16周龄MRL/lpr小鼠，对照组注射生理盐水。在小鼠20周龄时将其处死，取外周血、肾脏、长骨(股骨和胫骨)标本。肾脏标本固定、石蜡包埋，行HE、trichrome、periodic acid-schiff(pas)染色，观察肾小球基底膜功能异常恢复及肾小球膜细胞过度增长恢复情况。通过ELISA检测血清dsDNA-IgG,dsDNA-IgM，ANA水平，检测尿中和血清中补体3(complement3，C3)、肌酸酐(creatinine)、尿蛋白(urine protein)水平。 C57BL/6J and C3MRL-Fas lpr /J (MRL/lpr) mice (female, 6–7 weeks old), Beige nude/nude Xid(III) mice (female, 8–12 weeks old) were provided by . This experiment was approved by the Ethics Committee of Capital Medical University. The experiment was divided into the following three groups: 1. Blank control group (n=3), without any treatment. 2. BMMSCs transplantation group (n=3). 3. Transplant SHED group (n=3). Under general anesthesia, SHED or BMMSCs (1x10 5 cells/10g body weight, cells suspended in 100ml PBS) were injected into 16-week-old MRL/lpr mice through the tail vein, and the control group was injected with normal saline. The mice were sacrificed at the age of 20 weeks, and samples of peripheral blood, kidney, and long bones (femur and tibia) were collected. Kidney specimens were fixed, embedded in paraffin, and stained with HE, trichrome, and periodic acid-schiff (pas) to observe the abnormal recovery of glomerular basement membrane function and excessive growth of mesangial cells. Serum dsDNA-IgG, dsDNA-IgM, and ANA levels were detected by ELISA, and levels of complement 3 (complement 3, C3), creatinine, and urine protein in urine and serum were detected.
2)结果和说明2) Results and description
SLE是以多脏器受累和血中存在多种抗体为特征的自身免疫病，许多研究证实T、B淋巴细胞过度活化是SLE发病中的关键环节。T、B淋巴细胞来源于淋巴干/祖细胞，而MSCs在淋巴干/祖细胞增殖分化中具有重要的作用，所以MSCs在SLE致病中的作用越来越受到关注。SLE患者MSCs存在异常，它分化的骨髓基质细胞不能较好地支持淋巴细胞生长，淋巴细胞的生长依赖于淋巴细胞本身和骨髓基质细胞分泌的各种细胞因子，骨髓基质细胞的异常参与了淋巴细胞的异常活化。Majumdar等研究表明MSCs表达大量的细胞黏附分子，在细胞间黏附、归巢、支持造血、调节免疫细胞功能中起重要作用。当把肿瘤细胞种植人异体小鼠时，肿瘤细胞会被宿主免疫系统清除，而当肿瘤细胞和MSCs同时注入异体小鼠后，肿瘤细胞可在受体内存活，表明MSCs在体内具有免疫抑制功能。MSCs在分化成其他细胞类型时仍保留其免疫调节作用，这就意味着移植的MSCs可发挥长期的免疫调节作用。 SLE is an autoimmune disease characterized by the involvement of multiple organs and the presence of various antibodies in the blood. Many studies have confirmed that the overactivation of T and B lymphocytes is the key link in the pathogenesis of SLE. T and B lymphocytes are derived from lymphoid stem/progenitor cells, and MSCs play an important role in the proliferation and differentiation of lymphoid stem/progenitor cells, so the role of MSCs in the pathogenesis of SLE has attracted more and more attention. There are abnormalities in MSCs in patients with SLE. The differentiated bone marrow stromal cells cannot well support the growth of lymphocytes. The growth of lymphocytes depends on various cytokines secreted by lymphocytes themselves and bone marrow stromal cells. The abnormality of bone marrow stromal cells participates in the development of lymphocytes. abnormal activation. Studies by Majumdar et al. have shown that MSCs express a large number of cell adhesion molecules, which play an important role in intercellular adhesion, homing, supporting hematopoiesis, and regulating immune cell functions. When transplanting tumor cells into human allogeneic mice, the tumor cells will be cleared by the host immune system, and when tumor cells and MSCs are injected into the allogeneic mice at the same time, the tumor cells can survive in the recipient, indicating that MSCs have an immunosuppressive function in vivo . MSCs retain their immunomodulatory effects when differentiated into other cell types, implying that transplanted MSCs can exert long-term immunomodulatory effects. the
体外实验证实SHED具有MSCs生物学及免疫学特性，可以与T淋巴细胞相互影响。与BMMSCs相比，SHED通过恢复外周血Tregs/Th17比例，减少Th17细胞数目发挥免疫学效应。16周龄的MRL/lpr小鼠注射SHED后，可以发现小梁骨再生，破骨细胞活性受到了抑制。注射SHED的小鼠与注射BMMSCs的小鼠一样，重建了成骨细胞微环境，从而改善了其功能紊乱。MRL/lpr小鼠注射SHED后，虽说Treg水平未提高，但Treg/Th17细胞比例明显增高，这表明SHED的免疫调节功能可能源于Treg抑制自身免疫而Th17则可促进自身免疫 和炎症。 In vitro experiments confirmed that SHED has the biological and immunological characteristics of MSCs, and can interact with T lymphocytes. Compared with BMMSCs, SHED exerts an immunological effect by restoring the Tregs/Th17 ratio in peripheral blood and reducing the number of Th17 cells. After 16-week-old MRL/lpr mice were injected with SHED, trabecular bone regeneration and osteoclast activity were inhibited. Mice injected with SHED reconstituted the osteoblast microenvironment and thereby ameliorated its dysfunction just like mice injected with BMMSCs. After MRL/lpr mice were injected with SHED, although the level of Treg did not increase, the ratio of Treg/Th17 cells increased significantly, which indicated that the immune regulation function of SHED may be due to the suppression of autoimmunity by Treg and the promotion of autoimmunity and inflammation by Th17. the
在本实施例方法中，提供的一个实验结果见图11所示，其中详细描绘了脱落乳牙牙髓干细胞治疗系统性红斑狼疮鼠血清及肾脏组织学改变的情况。从图中结果可见本实施例实现了本发明的目的。 In the method of this embodiment, an experimental result provided is shown in FIG. 11 , which depicts in detail the histological changes in serum and kidney of rats with systemic lupus erythematosus treated with dental pulp stem cells from exfoliated deciduous teeth. It can be seen from the results in the figure that this embodiment has achieved the object of the present invention. the
总之，本发明表明，从脱落的乳牙牙髓可提取出SHED，它是来源于中胚层的间充质干细胞，具有MSCs的生物学和免疫学特性及功能。尽管SHED的免疫调节作用机制尚未明确，但由于它的免疫调节作用，在自身免疫病方面有潜在的治疗作用，SHED移植可作为治疗自身免疫病的新尝试。 In conclusion, the present invention shows that SHED can be extracted from the pulp of exfoliated deciduous teeth. It is a mesenchymal stem cell derived from mesoderm and has the biological and immunological characteristics and functions of MSCs. Although the immunomodulatory mechanism of SHED has not been clarified yet, due to its immunomodulatory effect, it has a potential therapeutic effect in autoimmune diseases, and SHED transplantation can be used as a new attempt to treat autoimmune diseases. the
实施例5：同种异体人牙周膜干细胞(hPDLSCs)用于治疗牙周病的标准操作实例Example 5: The standard operation example of allogeneic human periodontal ligament stem cells (hPDLSCs) for the treatment of periodontal disease
本实施例参考图12来说明同种异体人牙周膜干细胞(hPDLSCs)用于治疗牙周病的标准操作的一个实例： This embodiment refers to Figure 12 to illustrate an example of the standard operation of allogeneic human periodontal ligament stem cells (hPDLSCs) for the treatment of periodontal disease:
(a)采集人牙周膜(periodontal ligament，PDL)。从18-28岁患者采集正常的阻生第三磨牙(impacted third molar)。将PDL轻轻地从牙表面分离。 (a) Human periodontal ligament (PDL) was collected. Normal impacted third molars were collected from patients aged 18-28 years. Gently detach the PDL from the tooth surface. the
(b)培养hPDLSCs。分离hPDLSCs。原始培养15天后，来自一个阻生第三磨牙的hPDLSCs的数量约为4.90±0.34×105(P0;n=10)。再培养另外15天后，hPDLSCs的数量(P3)增加到8.86±0.46×106(n=10)。hPDLSCs对CD146和CD90显阳性。 (b) Culture of hPDLSCs. Isolation of hPDLSCs. After 15 days of primary culture, the number of hPDLSCs from one impacted third molar was about 4.90±0.34×10 5 (P0;n=10). After another 15 days of culture, the number of hPDLSCs (P3) increased to 8.86±0.46×10 6 (n=10). hPDLSCs were positive for CD146 and CD90.
(c)冷冻保存hPDLSCs。将第三代hPDLSCs用10%DMSO和90%FBS冷冻保存，并保存在液氮中。 (c) Cryopreservation of hPDLSCs. The third passage hPDLSCs were cryopreserved with 10% DMSO and 90% FBS and kept in liquid nitrogen. the
(d)融化hPDLSCs。融化之后，对hPDLSCs检查支原体、细菌、集落形成效率、间质干细胞标记模式和核型分析。 (d) hPDLSCs were thawed. After thawing, hPDLSCs were checked for mycoplasma, bacteria, colony formation efficiency, mesenchymal stem cell marker pattern and karyotype analysis. the
(e)制备hPDLSCs片。在100mm培养皿中对三种不同细胞数(1×105,1×106和2×106;n=3)培养12-15天，在1×106和2×106组中形成细胞片，但在1×105组中未形成细胞片。为此，将1×106的hPDLSCs接种到100mm培养皿中，达15天。 (e) Preparation of hPDLSCs sheets. Three different cell numbers (1×10 5 , 1×10 6 and 2×10 6 ; n=3) were cultured for 12-15 days in a 100 mm dish, forming in groups of 1×10 6 and 2×10 6 Cell sheets, but no cell sheets were formed in the 1×10 5 group. For this, 1 × 106 hPDLSCs were seeded into 100 mm Petri dishes for 15 days.
(f)然后，将40mg HA/TCP置于这些培养皿中。 (f) Then, 40 mg HA/TCP was placed in these Petri dishes. the
(g)hPDLSCs片与HA/TCP的全图。 (g) Full image of hPDLSCs sheet with HA/TCP. the
(h)口腔牙周损伤图。 (h) Diagram of oral periodontal injury. the
在牙周初始治疗(i)之后，将两个同种异体hPDLSCs片与HA/TCP植入到3mm×5mm×7mm的牙周损伤缺陷处(j)。 After initial periodontal treatment (i), two allogeneic hPDLSCs sheets were implanted with HA/TCP into a 3 mm × 5 mm × 7 mm periodontal lesion defect (j). the
(k)接下来的项目包括临床和放射照相评价、血液学和免疫学评价。 (k) Subsequent items include clinical and radiographic evaluation, hematology and immunology evaluation. the
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104232568A (en) * | 2014-08-29 | 2014-12-24 | 丁刚 | Osteogenic induction method of periodontal ligament stem cells (PDLSCs) |
| CN106011053A (en) * | 2016-06-27 | 2016-10-12 | 安徽新生命干细胞科技有限公司 | Novel dental pulp stem cell culturing medium |
| JP2017507123A (en) * | 2014-01-27 | 2017-03-16 | ベイジン ストマトロジー ホスピタル，キャピタル メディカル ユニバーシティ | Use of odontogenic stem cells and genetically modified odontogenic stem cells |
| CN106544316A (en) * | 2016-10-18 | 2017-03-29 | 丁刚 | A kind of preparation method of dental pulp stem cell diaphragm |
| CN106890363A (en) * | 2015-12-17 | 2017-06-27 | 西安组织工程与再生医学研究所 | A kind of preparation method for being engineered dental pulp |
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| CN102755667B (en) * | 2012-07-05 | 2014-06-25 | 中国人民解放军第四军医大学 | Preparation method and application of tissue-engineering human tooth root implantation material |
| CN102807967A (en) * | 2012-08-31 | 2012-12-05 | 沙船（天津）生物科技发展有限公司 | Application of deciduous tooth pulp to preparation of mesenchymal stem cell and culturing method of deciduous tooth pulp mesenchymal stem cell |
| CN112481364B (en) * | 2020-12-07 | 2023-03-24 | 上海交通大学医学院附属第九人民医院 | Method for screening new sub-population of periodontal ligament specific stem cells based on single cell sequencing and application thereof |
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| WO2008067488A2 (en) * | 2006-11-29 | 2008-06-05 | University Of Southern California | Mesenchymal stem cell-mediated functional tooth regeneration |
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| AU2003231015B2 (en) * | 2003-04-19 | 2009-10-08 | Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services National Institutes Of Health | Postnatal stem cells and uses thereof |
| AU2004293841B2 (en) * | 2003-11-20 | 2009-10-08 | Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Multipotent postnatal stem cells from human periodontal ligament and uses thereof |
| CN1587393A (en) * | 2004-07-15 | 2005-03-02 | 福建师范大学 | Technology for tooth regeneration using keratin stem cell and dental pulp stem cell |
| CN1896228A (en) * | 2005-07-12 | 2007-01-17 | 叶庆良 | A kind of dental stem cell and preparation method thereof |
| CN101144069A (en) * | 2007-08-22 | 2008-03-19 | 黄显成 | Method for culturing mesenchymal stem cells from oral tissue |
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| WO2008067488A2 (en) * | 2006-11-29 | 2008-06-05 | University Of Southern California | Mesenchymal stem cell-mediated functional tooth regeneration |
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| MASAKO MIURA等: "SHED: Stem cells from human exfoliated deciduous teeth", 《 PNAS》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2017507123A (en) * | 2014-01-27 | 2017-03-16 | ベイジン ストマトロジー ホスピタル，キャピタル メディカル ユニバーシティ | Use of odontogenic stem cells and genetically modified odontogenic stem cells |
| US10000738B2 (en) | 2014-01-27 | 2018-06-19 | Beijing Stomatology Hospital, Capital Medical University | Usage of odontogenic stem cells and genetically modified odontogenic stem cells |
| CN104232568A (en) * | 2014-08-29 | 2014-12-24 | 丁刚 | Osteogenic induction method of periodontal ligament stem cells (PDLSCs) |
| CN106890363A (en) * | 2015-12-17 | 2017-06-27 | 西安组织工程与再生医学研究所 | A kind of preparation method for being engineered dental pulp |
| CN106890363B (en) * | 2015-12-17 | 2021-09-10 | 西安组织工程与再生医学研究所 | Preparation method of engineered dental pulp |
| CN106011053A (en) * | 2016-06-27 | 2016-10-12 | 安徽新生命干细胞科技有限公司 | Novel dental pulp stem cell culturing medium |
| CN106544316A (en) * | 2016-10-18 | 2017-03-29 | 丁刚 | A kind of preparation method of dental pulp stem cell diaphragm |
| CN113662968A (en) * | 2021-09-18 | 2021-11-19 | 哈尔滨科技实业开发有限公司 | A pharmaceutical composition for preventing or treating erectile dysfunction |
| CN113662968B (en) * | 2021-09-18 | 2023-09-19 | 哈尔滨科技实业开发有限公司 | Pharmaceutical composition for treating erectile dysfunction |
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| CN102292435B (en) | 2013-08-07 |
| WO2010083730A1 (en) | 2010-07-29 |
| CN102292435A (en) | 2011-12-21 |
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