CN106957821B - 一种调控间充质干细胞定向分化的方法 - Google Patents
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Abstract
本发明公开了一种调控间充质干细胞定向分化方法。该方法可以促进间充质干细胞向血管内皮细胞分化，抑制间充质干细胞向血管平滑肌细胞分化。本发明技术方案为：合成目的基因片段HMGB1 cDNA，将目的基因片段插入慢病毒质粒基因组，所述慢病毒质粒基因组含有嘌呤霉素抗性基因和荧光蛋白基因片段；将所述慢病毒转染间充质干细胞，经嘌呤霉素筛选获得阳性转染细胞，经蛋白定量检测确定细胞HMGB1表达上调。采用HMGB1表达上调的方法可以促进间充质干细胞在VEGF诱导下向血管内皮细胞分化，抑制间充质干细胞在PDGF诱导下向血管平滑肌细胞分化。
Description
技术领域
本发明属于干细胞技术领域，具体涉及一种调控间充质干细胞定向分化的方法。
背景技术
血管内皮细胞和血管平滑肌细胞是构成血管壁的两种主要血管细胞，它们都可以由间充质干细胞分化而来。间充质干细胞是一种来源于中胚层具有自我复制力和多向分化潜能的成体干细胞，可以分化成脂肪细胞、软骨细胞、骨细胞和血管细胞等多种成体细胞。间充质干细胞分布于全身结缔组织和器官间质中，生理状态下数量稀少且处于静息休眠状态，受到刺激后迁移至病变部位(也叫做归巢)，分化为成体细胞。
VEGF(vascular endothelial growth factor)是诱导干细胞向血管内皮细胞分化的重要细胞因子[1]，其结构上由两条同源肽链通过二硫键连接而成。VEGF包括7个亚型，其中VEGF-A应用最广泛，VEGF-A有5种异构体，它们都能诱导血管内皮细胞增殖。VEGFR(VEGF receptor)属于酪氨酸激酶受体，包括三种亚型，其中VEGFR-2主要介导血管内皮细胞的有丝分裂、存活和通透性，被认为是血管新生的一种标志[2]。VEGF是一种重要的促有丝分裂原，能够促进血管内皮细胞分裂、增殖、迁移和趋化[3]。
PDGF(platelet-derived growth factor)是诱导干细胞向血管平滑肌细胞分化的重要细胞因子[1]，其结构上由两条肽链通过二硫键连接而成，这些肽链包括A、B、C和D四种，这使得PDGF具有多种亚型，即为PDGF-AA、PDGF-BB、PDGF-AB、PDGF-CC以及PDGF-DD。PDGFR(PDGF receptor)由α和β两个亚单位构成，它们与不同亚型PDGF的亲和力差别很大，PDGF-AA和PDGF-CC选择性结合PDGFRα亚单位，PDGF-BB和PDGF-AB与PDGFRα和β亚单位都可以结合，PDGF-DD只结合PDGFRβ亚单位。PDGF是一种重要的促有丝分裂原，可以诱导血管平滑肌细胞表型转化，从收缩型转化成分泌型，促进细胞增殖和迁移[4]。
虽然，目前间充质干细胞的分化机制和诱导分化的信号通路还不十分清楚，但是间充质干细胞所处的微环境是决定其分化的重要因素[5]。在复杂环境下(如VEGF和PDGF等多种诱导分化因子同时存在的条件下[6,7])，间充质干细胞的分化倾向确实难以判断。此时，如何采取有效的方法调控干细胞定向分化是本领域的一项技术难题。
HMGB1(highmobility groupbox 1)是一种高度保守的小分子核蛋白，结构上包括Abox和B box两个结构域，B box是HMGB1发挥促进炎症反应作用的功能区域，Abox具有抗炎作用，是抑制HMGB1诱发炎症反应的拮抗剂。Abox和B box都能够与DNA结合，并参与DNA双链的构象改变。HMGB1还是一种损伤相关分子模式(damage associated molecule pattern)分子，当组织受损时细胞核内HMGB1赖氨酸残基发生乙酰化修饰，促使HMGB1由细胞核转位至细胞质，随后被释放到细胞外传递损伤信号。此外，损伤引起的细胞死亡(necrosis)可以直接释放大量HMGB1至细胞外。细胞外HMGB1与细胞膜受体结合，通过激活MAPK(mitogen-activated protein kinase)和NF-κB(nuclear factor kappa-light-chain-enhancerofactivated B cells)等下游信号通路，诱发炎症反应和免疫应答[8]。
前人研究发现HMGB1可以作为趋化因子促进间充质干细胞归巢[9,10]，HMGB1过表达的间充质干细胞可以分泌细胞因子VEGF和PCNA(proliferating cell nuclear antigen)[11]。但是，前人研究不涉及HMGB1在间充质干细胞向血管内皮细胞分化中的调控作用，也不涉及HMGB1在间充质干细胞向血管平滑肌细胞分化中的调控作用。以上涉及的参考文献如下：
[1]Patsch C,Challet-Meylan L,Thoma EC,et al.Generation ofvascularendothelial and smooth muscle cells from human pluripotent stem cells[J].NatCell Biol,2015,17(8):994-1003.
[2]Kajdaniuk D,Marek B,Borgiel-Marek H,et al.Vascular endothelialgrowth factor(VEGF)-part 1:inphysiology andpathophysiology[J].Endokrynol Pol,2011,62(5):444-455.
[3]Wu X,Zhao Y,Tang C,et al.Re-endothelialization study onendovascular stents seeded by endothelial cells through up-or downregulationof VEGF[J].ACS Appl Mater Interfaces,2016,8(11):7578-7589.
[4]Spin JM,Maegdefessel L,Tsao PS.Vascular smooth muscle cellphenotypic plasticity:focus on chromatin remodelling[J].Cardiovasc Res,2012,95(2):147-155.
[5]刘佳佳,张亦婷,彭航,等.间充质干细胞在动脉粥样硬化治疗中的研究进展[J].生物工程学报,2013,29(11):1538-1547.
[6]Nykanen AI,Krebs R,Tikkanen JM,et al.Combined vascular endothelialgrowth factor and platelet-derived growth factor inhibition in rat cardiacallografts:beneficial effects on inflammation and smooth muscle cellproliferation[J].Transplantation,2005,79(2):182-189.
[7]Cagnin S,Biscuola M,Patuzzo C,et al.Reconstruction and functionalanalysis of altered molecularpathways in human atherosclerotic arteries[J].BMC Genomics,2009,10:13.
[8]Huebener P,Hernandez C,Schwabe RF.HMGB1and injury amplification[J].Oncotarget,2015,6(27):23048-23049.
[9]Lotfi R,Eisenbacher J,Solgi G,et al.Human mesenchymal stem cellsrespond to native but not oxidized damage associated molecular patternmolecules from necrotic(tumor)material[J].Eur J Immunol,2011,41(7):2021-2028.
[10]Xie HL,Zhang Y,Huang YZ,et al.Regulation of high mobility groupbox 1 and hypoxia in the migration ofmesenchymal stem cells[J].Cell Biol Int,2014,38(7):892-897.
[11]Niu S,Jian L,Zhang L.Protective effects of mesenchymal stem cellswith transient overexpression of hmgbl on balloon-induced carotid arteryinjury[J].European Journal of Inflammation,2012,10(3):347-356.
发明内容
针对现有技术中存在的不足，本发明所要解决的技术问题是提供一种调控间充质干细胞定向分化的方法，该方法可以促进间充质干细胞向血管内皮细胞分化，同时又能抑制间充质干细胞向血管平滑肌细胞分化。
本发明的目的是通过如下技术方案实现的：
一种调控间充质干细胞定向分化的方法，具体为：上调间充质干细胞中HMGB1的表达。
所述定向分化的分化方向为：促进间充质干细胞向血管内皮细胞分化，抑制间充质干细胞向血管平滑肌细胞分化。
所述定向分化的方法为：利用携带表达所述HMGB1基因的慢病毒转染间充质干细胞，促进间充质干细胞中HMGB1表达上调。
所述携带表达HMGB1基因的慢病毒的构建过程为：采用Primer-blast软件设计两条引物用于RT-PCR扩增HMGB1cDNA，两端插入Flag蛋白编码序列、终止序列及限制性酶切识别位点，取合成的寡核苷酸片段，退火，在T4DNA连接酶作用下，连接入经限制性酶切线性化的慢病毒穿梭质粒，获得连接产物pLVTH-HMGB1-GFP-PURO，转化感受态DH-5α大肠杆菌，经卡那霉素筛选扩增后，利用质粒小量提取试剂盒提取纯化质粒，酶切后测序鉴定，证实穿梭质粒中插入片段的碱基序列与设计序列完全一致，并设阴性对照重组质粒；取慢病毒穿梭质粒pLVTH-HMGB1-GFP-PURO、包装质粒pCMV-dR8.2dvpr和包膜质粒pCMV-VSV-G共同转染293T细胞，包装获得携带HMGB1基因的慢病毒质粒，即pLV-HMGB1；该病毒质粒中还含有嘌呤霉素抗性基因(PURO)和绿色荧光蛋白基因(GFP)，与HMGB1基因在同一个启动子调控下实现共同表达；阴性对照病毒(pLV-control)不含HMGB1cDNA，但保留嘌呤霉素抗性基因和绿色荧光蛋白基因。
所述转染间充质干细胞的方法为：将所述慢病毒转染间充质干细胞，经嘌呤霉素筛选获得阳性转染的细胞，经蛋白定量检测确定细胞HMGB1表达上调。
更进一步地，所述转染间充质干细胞的方法为：将间充质干细胞贴壁培养在DMEM培养液中，培养液中添加了10％胎牛血清和青霉素-链霉素抗菌液。将间充质干细胞接种于六孔培养板中，每孔含2×105个细胞，置于二氧化碳培养箱中培养24小时，加入pLV-HMGB1(阴性对照组加入pLV-control)，再培养8小时后更换新鲜培养液继续培养，培养第4天加入嘌呤霉素筛选阳性转染克隆，借助免疫荧光显微镜观察阳性转染细胞(发绿色荧光)。使用Western blot检测间充质干细胞的蛋白表达情况，检测HMGB1表达所用一抗为兔抗大鼠HMGB1抗体，二抗为HRP标记山羊抗兔抗体，通过病毒转染过表达的HMGB1含有Flag蛋白标记，可以用抗Flag抗体检测，所用一抗为小鼠抗Flag抗体，二抗为HRP标记山羊抗小鼠抗体。
本发明采用上调HMGB1表达的方法，获得以下有益效果：
采用HMGB1表达上调方法改变间充质干细胞的分化倾向，促进间充质干细胞在VEGF诱导下向血管内皮细胞分化，抑制间充质干细胞在PDGF诱导下向血管平滑肌细胞分化。
附图说明
图1-1：pLV-control转染的间充质干细胞表达GFP(发绿色荧光)；
图1-2：pLV-HMGB1转染的间充质干细胞表达GFP(发绿色荧光)；
图1-3：Western blot检测pLV-control和pLV-HMGB1转染的间充质干细胞HMGB1表达情况；
图1-4：Western blot检测pLV-HMGB1转染的间充质干细胞表达有Flag标记的HMGB1；
图2-1：流式细胞术检测pLV-control和pLV-HMGB1转染的间充质干细胞向CD31表达阳性细胞分化情况；
图2-2：流式细胞术检测pLV-control和pLV-HMGB1转染的间充质干细胞向αSMA表达阳性细胞分化情况；
图3-1：荧光显微镜下检查pLV-control大鼠移植动脉新生血管内膜GFP标记细胞(发绿色荧光)；
图3-2：荧光显微镜下检查pLV-control大鼠移植动脉新生血管内膜CD31表达阳性细胞(发红色荧光)；
图3-3：荧光显微镜下检查pLV-HMGB1大鼠移植动脉新生血管内膜GFP标记细胞(发绿色荧光)；
图3-4：荧光显微镜下检查pLV-HMGB1大鼠移植动脉新生血管内膜CD31表达阳性细胞(发红色荧光)；
图3-5：荧光显微镜下检查pLV-control大鼠移植动脉新生血管内膜GFP标记细胞(发绿色荧光)；
图3-6：荧光显微镜下检查pLV-control大鼠移植动脉新生血管内膜αSMA表达阳性细胞(发红色荧光)；
图3-7：荧光显微镜下检查pLV-HMGB1大鼠移植动脉新生血管内膜GFP标记细胞(发绿色荧光)；
图3-8：荧光显微镜下检查pLV-HMGB1大鼠移植动脉新生血管内膜αSMA表达阳性细胞(发红色荧光)。
具体实施方式
下面将结合本发明实施例，对本发明采用的调控方法进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：制备HMGB1过表达的间充质干细胞
1、材料和试剂，见下表1：
表1
2、方法：
病毒构建过程：Primer-blast软件设计两条引物用于RT-PCR扩增HMGB1 cDNA，两端插入Flag蛋白编码序列、终止序列及限制性酶切识别位点，取合成的寡核苷酸片段，缓慢退火，在T4DNA连接酶作用下，连接入经限制性酶切线性化的慢病毒穿梭质粒，获得连接产物pLVTH-HMGB1-GFP-PURO，转化感受态DH-5α大肠杆菌，经卡那霉素筛选扩增后，利用质粒小量提取试剂盒提取纯化质粒，酶切后测序鉴定，证实穿梭质粒中插入片段的碱基序列与设计序列完全一致，并设阴性对照重组质粒。取慢病毒穿梭质粒pLVTH-HMGB1-GFP-PURO、包装质粒pCMV-dR8.2dvpr和包膜质粒pCMV-VSV-G共同转染293T细胞，包装获得携带HMGB1基因的慢病毒质粒(pLV-HMGB1)。该病毒质粒中还含有嘌呤霉素抗性基因(PURO)和绿色荧光蛋白基因(GFP)，与HMGB1基因在同一个启动子调控下实现共同表达。阴性对照病毒(pLV-control)不含HMGB1cDNA，但保留嘌呤霉素抗性基因和绿色荧光蛋白基因。
病毒转染过程：间充质干细胞贴壁培养在DMEM培养液中，培养液中添加了10％胎牛血清和青霉素-链霉素抗菌液。将间充质干细胞接种于六孔培养板中，每孔含2×105个细胞，置于二氧化碳培养箱中培养24小时，加入pLV-HMGB1(阴性对照组加入pLV-control)，再培养8小时后更换新鲜培养液继续培养，培养第4天加入嘌呤霉素筛选阳性转染克隆，借助免疫荧光显微镜观察阳性转染细胞(发绿色荧光)。使用Western blot检测间充质干细胞的蛋白表达情况，检测HMGB1表达所用一抗为兔抗大鼠HMGB1抗体，二抗为HRP标记山羊抗兔抗体，通过病毒转染过表达的HMGB1含有Flag蛋白标记，可以用抗Flag抗体检测，所用一抗为小鼠抗Flag抗体，二抗为HRP标记山羊抗小鼠抗体。
3、结果：病毒转染后大于99.9％间充质干细胞表达GFP，发绿色荧光(见图1-1和图1-2)，pLV-HMGB1转染的间充质干细胞HMGB1表达水平比pLV-control显著升高(图1-3)，检测到pLV-HMGB1转染的间充质干细胞表达含有Flag蛋白标记的HMGB1(图1-4)。由此表明采用病毒转染的方法，间充质干细胞实现了HMGB1表达上调。
实施例2：实施例1中HMGB1过表达的间充质干细胞体外分化能力的评价
1、材料和试剂，见下表2：
表2
2、方法：培养液中加入VEGF诱导间充质干细胞向血管内皮细胞分化，VEGF在培养液中浓度为25ng/ml，体外培养14天；培养液中加入PDGF诱导间充质干细胞向血管平滑肌细胞分化，PDGF在培养液中浓度为12.5ng/ml，体外培养14天。
采用流式细胞术检测分化结果。CD31是血管内皮细胞特异性蛋白标记，流式细胞术中用PE标记CD31抗体检测CD31表达阳性细胞，细胞CD31表达阳性提示成功分化成血管内皮细胞。αSMA是血管平滑肌细胞特异性蛋白标记，流式细胞术中用PE标记αSMA抗体检测αSMA表达阳性细胞，细胞αSMA表达阳性提示成功分化成血管平滑肌细胞。
3、结果：pLV-HMGB1转染的间充质干细胞分化成CD31表达阳性细胞的比例比pLV-control增加(图2-1，图中竖线表示使用同型对照抗体设置的阴性阈值)，分化成αSMA表达阳性细胞的比例比pLV-control降低(图2-2，图中竖线表示使用同型对照抗体设置的阴性阈值)。由此可见上调HMGB1表达可以促进间充质干细胞向血管内皮细胞分化，抑制间充质干细胞向血管平滑肌细胞分化。
实施例3：实施例1中HMGB1过表达的间充质干细胞体内分化能力的评价
1、实验动物，见下表3：
表3
2、方法：
构建移植动脉硬化模型：F344和LEWIS都是近交系大鼠，它们的基因型比较接近，主要组织相容性复合体(MHC)位点基本一样，只是非MHC上部分不同，因此，这两个品系大鼠之间移植的腹主动脉可以发生慢性排斥反应，在病理形态学上表现为移植动脉硬化。首先获取LEWIS大鼠的腹主动脉作为移植物，然后取F344大鼠为受体鼠，阻断血流后剪断腹主动脉，将LEWIS大鼠的腹主动脉桥接在两侧断端之间，恢复腹主动脉通畅性。术后90天发现移植入腹主动脉发生动脉硬化。
动物分组和处理方式：取移植动脉硬化模型大鼠24只，分成两组，每组8只，详见表4。两组大鼠从移植术后30天开始以尾静脉注射的方式分别接种实施例1中两种慢病毒转染的间充质干细胞，每次接种细胞量2×106个细胞，每15天接种一次，共4次。
表4.动物实验分组
术后90天取大鼠移植动脉制作冰冻切片，利用免疫荧光技术标记CD31表达阳性细胞和αSMA表达阳性细胞，分析比对归巢细胞(发绿色荧光)表达CD31和αSMA情况。
3、结果：pLV-HMGB1组大鼠的新生血管内膜中归巢细胞表达CD31(图3-3和图3-4是同一视野，注意箭头标记的细胞)的比例比pLV-control组升高(图3-1和图3-2是同一视野，注意箭头标记的细胞)，pLV-HMGB1组大鼠的新生血管内膜中归巢细胞表达αSMA(图3-8和图3-7是同一视野，注意箭头标记的细胞)的比例比pLV-control组降低(图3-5和图3-6是同一视野，注意箭头标记的细胞)降低。由此可见HMGB1可以促进移植动脉新生血管内膜中归巢细胞向血管内皮细胞分化，抑制其向血管平滑肌细胞分化。
Claims (1)
1.一种调控间充质干细胞定向分化的方法，其特征在于：上调间充质干细胞中HMGB1的表达；
所述定向分化的方向为：促进间充质干细胞向血管内皮细胞分化，抑制间充质干细胞向血管平滑肌细胞分化；
利用携带表达所述HMGB1基因的慢病毒转染间充质干细胞，以促进所述间充质干细胞中HMGB1表达上调；
所述携带表达HMGB1基因的慢病毒的构建过程为：采用Primer-blast软件设计两条引物用于RT-PCR扩增HMGB1 cDNA，两端插入Flag蛋白编码序列、终止序列及限制性酶切识别位点，取合成的寡核苷酸片段，退火，在T4DNA连接酶作用下，连接入经限制性酶切线性化的慢病毒穿梭质粒，获得连接产物pLVTH-HMGB1-GFP-PURO，转化感受态DH-5α大肠杆菌，经卡那霉素筛选扩增后，利用质粒小量提取试剂盒提取纯化质粒，酶切后测序鉴定，证实穿梭质粒中插入片段的碱基序列与设计序列完全一致，并设阴性对照重组质粒；取慢病毒穿梭质粒pLVTH-HMGB1-GFP-PURO、包装质粒pCMV-dR8.2dvpr和包膜质粒pCMV-VSV-G共同转染293T细胞，包装获得携带HMGB1基因的慢病毒质粒，即为pLV-HMGB1；该病毒质粒中还含有嘌呤霉素抗性基因，即为PURO，和绿色荧光蛋白基因，即为GFP，与HMGB1基因在同一个启动子调控下实现共同表达；阴性对照病毒，即为pLV-control，不含HMGB1 cDNA，但保留嘌呤霉素抗性基因和绿色荧光蛋白基因；
所述转染间充质干细胞的方法为：将所述慢病毒转染间充质干细胞，经嘌呤霉素筛选获得转染的阳性细胞，经蛋白定量检测确定细胞HMGB1表达上调；
所述转染间充质干细胞的方法为：将间充质干细胞贴壁培养在DMEM培养液中，培养液中添加了10％胎牛血清和青霉素-链霉素抗菌液；将间充质干细胞接种于六孔培养板中，每孔含2×105个细胞，置于二氧化碳培养箱中培养24小时，加入pLV-HMGB1，而阴性对照组加入pLV-control，再培养8小时后更换新鲜培养液继续培养，培养第4天加入嘌呤霉素筛选阳性转染克隆，借助免疫荧光显微镜观察发绿色荧光的阳性转染细胞；使用Western blot检测间充质干细胞的蛋白表达情况，检测HMGB1表达所用一抗为兔抗大鼠HMGB1抗体，二抗为HRP标记山羊抗兔抗体，通过病毒转染过表达的HMGB1含有Flag蛋白标记，可以用抗Flag抗体检测，所用一抗为小鼠抗Flag抗体，二抗为HRP标记山羊抗小鼠抗体。
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