CN104215773B - A kind of method identifying mesenchymal stem cells MSCs activity - Google Patents
A kind of method identifying mesenchymal stem cells MSCs activity Download PDFInfo
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Abstract
The invention discloses a kind of method detecting GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs and the application in identifying mesenchymal stem cells MSCs activity thereof, the invention also discloses a kind of method identifying mesenchymal stem cells MSCs activity, the method comprises the following steps: 1) adopt cell immunofluorescent staining method detection GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs；2) if the GAPDH albumen substep of more than 60% is in nucleus, it is high expressed type, for high activity bone bone marrow-drived mesenchymal stem；If the GAPDH albumen substep of less than 40% is in nucleus, it is low expression type, for low activity mesenchymal stem cells MSCs.Present invention firstly discovers that the relation of GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs and mesenchymal stem cells MSCs activity, the method provided can identify rapidly disease therapeuticing effect is good, that multiplication capacity is strong mesenchymal stem cells MSCs, thus can be used for the high flux screening of mesenchymal stem cells MSCs.
Description
Technical field
The present invention relates to a kind of method detecting GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs and the application in identifying mesenchymal stem cells MSCs activity thereof, the invention still further relates to a kind of method identifying mesenchymal stem cells MSCs activity.
Background technology
Mesenchymal stem cells MSCs (BMSCs) is the non-hematopoietic stem cell that a group derives from myeloid tissue, belongs to pluripotent stem cell.Owing to BMSCs has Multidirectional Differentiation ability, can to various connective tissues and be derived partly from ectodermic histo-differentiation after cultivating in vitro, form adipose cell, myocyte, chondrocyte, neurocyte, endotheliocyte and osteoblast etc., therefore have wide application prospects at medical field.It has clear superiority compared with embryonic stem cell, is absent from the problems such as immunologic rejection, teratoma and ethics, is desirable tissue engineering seed cell, and it can make exogenous gene import and express, and is potential gene therapy target cell.
BMSCs used in stem-cell therapy its separate, concentration, purify, identify, detection, washing, the process operations process such as frozen complicated, few plus the content of BMSCs own, it is difficult to be made directly separation purification, and cell quantity there will be reduction owing to its growing environment changes to increase with the age, reduces the effect of stem-cell therapy all to a certain extent.Cytoactive declines not only can affect the therapeutic effect of disease, and can cause some side reactions, such as heating etc..If can in early days its quality be identified, only store and use the BMSCs that activity is high, just can improve safety and reduce cost.This needs a kind of method that can quickly differentiate that dry cell mass is good and bad, contributes to that the vigor that filters out is good, multiplication capacity strong, is more suitable for the BMSCs of disease treatment.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) participates in glucolytic a kind of key enzyme, is made up of the subunit of 4 30-40kDa.GAPDH gene almost in a organized way in all high level expressions, be widely used as the internal reference of Westernblot Protein Standardization.Because GAPDH as house-keeping gene allogenic cell or tissue in protein expression amount be usually constant.But, studies have found that the expression of GAPDH albumen in mesenchymal stem cells MSCs and non-constant, the applicant in conventional research it was unexpectedly observed that the high mesenchymal stem cells MSCs of GAPDH expressing quantity is better to the therapeutic effect of diseased n animal.Finding based on these, we expect the identification beacon using the expression of GAPDH albumen as mesenchymal stem cells MSCs activity.
Summary of the invention
First purpose of the present invention is to provide and a kind of detects GAPDH albumen method of expression and distribution level in mesenchymal stem cells MSCs.
Second purpose of the present invention is to provide said method application in identifying mesenchymal stem cells MSCs activity.
3rd purpose of the present invention is on the basis of described method, it is further provided a kind of method identifying mesenchymal stem cells MSCs activity.
Applicant adopts cell immunofluorescent staining method that the GAPDH protein expression level distribution in BMSCs is detected, and according to testing result, if the GAPDH albumen substep of 60% and more than 60% is in nucleus, is high expressed type；If the GAPDH albumen substep of less than 40% is in nucleus, it is low expression type.
Described cell immunofluorescent staining method is: by BMSCs through creep plate, embathe, fix, rupture of membranes, after closing, add GAPDH primary antibodie overnight incubation, gather image at fluorescence microscopy Microscopic observation after being subsequently adding that the fluorescence two of FITC labelling is anti-and hatching.
Applicant have further found that, close relationship is there is in GAPDH albumen expression and distribution level in BMSCs and BMSCs between the therapeutic effect of disease, especially in close relations with the therapeutic effect of pulmonary hypertension, its curative effect of BMSCs of high expressed GAPDH albumen apparently higher than low express GAPDH albumen BMSCs.
Applicant further found that, the competence for added value of BMSCs is also interfered significantly on by GAPDH albumen expression and distribution level in BMSCs, and the multiplication capacity of the BMSCs of high expressed GAPDH albumen is higher.
Therefore, detection GAPDH albumen method of expression and distribution level in mesenchymal stem cells MSCs provided by the invention can be used for identifying the activity of mesenchymal stem cells MSCs.
Invention further provides a kind of method identifying mesenchymal stem cells MSCs activity, it comprises the following steps:
1) cell immunofluorescent staining method detection GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs is adopted；
2) if the GAPDH albumen substep of more than 60% is in nucleus, it is high expressed type, for high activity bone bone marrow-drived mesenchymal stem；If the GAPDH albumen substep of less than 40% is in nucleus, it is low expression type, for low activity mesenchymal stem cells MSCs.
Present invention firstly discovers that the relation of GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs and mesenchymal stem cells MSCs activity, the method provided can identify rapidly disease therapeuticing effect is good, that multiplication capacity is strong mesenchymal stem cells MSCs, thus can be used for the high flux screening of mesenchymal stem cells MSCs.The mesenchymal stem cells MSCs of amplification in vitro each batch only need to take minimal amount of cell and carry out immunofluorescence operation, and the expression and distribution level of GAPDH in cell of analyzing just can determine whether the cell quality of this batch.Authentication method provided by the invention is without using large-scale instrument and equipment, it is not necessary to cell is carried out damaging operation, decreases the probability that mesenchymal stem cells MSCs sustains damage, pollutes, and also has that flow process is simple, easy and simple to handle, low cost and other advantages simultaneously.
Accompanying drawing explanation
Fig. 1 is the fluorescence microscope images of the BMSCs of the BMSCs of high expressed GAPDH albumen (H-GAPDH) and low expression GAPDH albumen (L-GAPDH).
Fig. 2 is the BMSCs of the different GAPDH expression impact that MCT induces the Right ventricular hypertrophy degree of pulmonary hypertension.
Fig. 3 is the BMSCs of the different GAPDH expression impact that MCT induces the blood vessel flesh degree of pulmonary hypertension.
Fig. 4 is the different GAPDH expression impacts on the multiplication capacity of BMSCs.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is described in detail, but should not be understood as limiting of its scope.
The activity identification of embodiment 1 mesenchymal stem cells MSCs
(1) Immunofluorescent localization GAPDH expression and distribution in BMSCs
By mesenchymal stem cells MSCs through creep plate, embathe, fix, rupture of membranes, after closing, add GAPDH primary antibodie overnight incubation, after being subsequently adding that the fluorescence two of FITC labelling is anti-and hatching, gather image at fluorescence microscopy Microscopic observation.Concrete operations are as follows:
First day:
1. in culture plate, the slide PBS having climbed mesenchymal stem cells MSCs is embathed 3 times, each 3min；
2. slide is embathed 3 times with the paraformaldehyde of 4% fixing creep plate 15min, PBS, each 3min；
3. 0.5%TritonX-100 (PBS preparation) the penetrating 20min of room temperature；
4. PBS embathes slide 3 times, each 3min, blots PBS, 5%BSA room temperature and closes 30min；
5. GAPDH primary antibodie (GAPDH monoclonal antibody is purchased from CellSignalingTechnology, article No. 2188), 4 DEG C of overnight incubation in wet box are added；
Second day:
6. the fluorescence two adding FITC labelling resists: PBS embathes creep plate 3 times, each 3min, blot and creep plate drips after surplus liquid the anti-(ZF-0311 of fluorescence two diluted, it is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge), incubated at room 1h in wet box, PBS embathes creep plate 3 times, each 3min；From add fluorescence two anti-, after all operations step all carry out in relatively dark place.
7. gather image at fluorescence microscopy Microscopic observation, see Fig. 1.
Cell density is not easily too high, cell density during for fixing proper below 50% (overstocked be likely to result in false negative and GAPDH be that dispersivity is distributed)；Fixing avoiding cell to dry for a long time before, the fixative ice methanol for fixing BMSCs is better than ice formaldehyde (after fixing cellular morphology closer to cultivation conditions)；Primary antibodie and two resists extremely important, should select highly sensitive monoclonal antibody as far as possible, otherwise be likely to occur false negative.The fluorescent sample that labelling is good should keep lucifuge, observes as early as possible, and otherwise false negative easily occurs in fluorescent quenching.If antibody diluted concentration is improper, then it is likely to occur false positive such as excessive concentration.
GAPDH concentration is expressed in nucleus (the GAPDH albumen substep of 60% and more than 60% is in nucleus) and is decided to be H-GAPDH, i.e. high expressed type (Figure 1A), for high-quality mesenchymal stem cells MSCs, such cytoactive and competence for added value are higher；And disperse to be expressed in cell cytosol and core (the GAPDH albumen substep of less than 40% is in nucleus), for L-GAPDH, i.e. low expression type (Figure 1B), for low quality mesenchymal stem cells MSCs, such cytoactive and competence for added value are poor.
The impact on pulmonary hypertension therapeutic effect of the embodiment 2GAPDH albumen expression and distribution level in BMSCs
1. pulmonary hypertension Animal Model
Carrying out, according to international standard, the operation that animal is relevant, select 180g male SD mouse, tail vein injection monocrotaline (MCT, 60mg/kg) sets up pulmonary hypertension model, 30 days altogether.Modeling the 4th day and start, cell therapy group injects the BMSCs (1 × 10 of H-GAPDH, L-GAPDH respectively through tail vein6Individual/0.5mLPBS), blank group injection 0.5mLPBS.Modeling put to death rat after 30 days, detected Pulmonic flesh and right ventricle plumpness situation.
2. the plump detection with pulmonary artery flesh of right ventricle
Separate right ventricle and left ventricle and interval, weigh respectively, record, then by the weight at right ventricle weight/left ventricle+interval, be used for evaluating right ventricle plumpness.After lung tissue is fixed with 4% paraformaldehyde, paraffin embedding, section, HE dyeing.Thin vessels 15-30 taking 25-100 μm of caliber in every induced lung measures its pipe thickness, with normal blank matched group blood vessel thickness for reference, the blood vessel thickness percentages calculating two kinds of different expression type bone mesenchymal stem cells treatment groups is pulmonary artery flesh rate.
3. experimental result
Result see Fig. 2,3 and table 1.
Right ventricular hypertrophy that table 1 is respectively organized and blood vessel flesh degree
* p < 0.05, vs. blank group；#p < 0.05, vs. monocrotaline process group；▲ p < 0.05, vs. high expressed GAPDH treatment group.
Blankcontrol is blank group；MCTtreatment is monocrotaline process group；MCT+H-GAPDH is GAPDH high expressed type stem-cell therapy group；MCT+L-GAPDH is the low expression type stem-cell therapy group of GAPDH.Result shows: BMSCs injection can alleviate pulmonary artery remodeling and the Right ventricular hypertrophy of MCT induction.The BMSCs (H-GAPDH) that GAPDH expression is high BMSCs (L-GAPDH) more remarkable effect that relatively GAPDH expression is low.
The impact on BMSCs multiplication capacity of the embodiment 3GAPDH albumen expression and distribution level in BMSCs
1.BMSCs multiplication capacity detects
Making single cell suspension after the BMSCs digestion of different expression types, after counting, be made into certain density single cell suspension and carry out plating cells, each sample sets 5～6 multiple holes, and edge hole adds sterilized water or PBS, 37 degree, 5%CO2Incubator is cultivated.CCK-8 process after 72h, microplate reader detects, according to data statistic analysis.
2. experimental result
As shown in Figure 4, the 24h rate of increase is organized under cell normal condition for 1 (1.005 ± 0.046 with high expressed GAPDH (H-GAPDH), n=7) it is standardized processing, the normal 24h propagation of the low GAPDH of expression (L-GAPDH) is (0.909 ± 0.054, n=8) of H-GAPDH；H-GAPDH cell is after 10 μMs of histamine stimulate 24h, and the rate of increase is (1.322 ± 0.027, n=8) under H-GAPDH normal condition；L-GAPDH cell is after 10 μMs of histamine stimulate 24h, and the rate of increase is (0.994 ± 0.032, n=8) under H-GAPDH normal condition.H-GAPDH cell and L-GAPDH cell 24h in normal state breed degree zero difference, and after histamine stimulates, H-GAPDH cell proliferation dramatically increases, and L-GAPDH cell proliferation is without significant change.
Claims (1)
1. the method identifying mesenchymal stem cells MSCs activity, it is characterised in that comprise the following steps:
1) cell immunofluorescent staining method detection GAPDH albumen expression and distribution level in mesenchymal stem cells MSCs is adopted；
2) if the GAPDH albumen of more than 60% is distributed in nucleus, it is high expressed type, for high activity bone bone marrow-drived mesenchymal stem；If the GAPDH albumen of less than 40% is distributed in nucleus, it is low expression type, for low activity mesenchymal stem cells MSCs,
Described cell immunofluorescent staining method is: by mesenchymal stem cells MSCs through creep plate, embathe, fix, rupture of membranes, after closing, add GAPDH primary antibodie overnight incubation, gather image at fluorescence microscopy Microscopic observation after being subsequently adding that the fluorescence two of FITC labelling is anti-and hatching.
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Citations (3)
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|---|---|---|---|---|
| WO2003043486A2 (en) * | 2001-11-16 | 2003-05-30 | Tufts University | Matrix for the production of tissue engineered ligaments, tendons and other tissue |
| WO2010101883A2 (en) * | 2009-03-06 | 2010-09-10 | Mayo Foundation For Medical Education And Research | Tendon or ligament tissue engineering |
| CN102533646A (en) * | 2011-12-27 | 2012-07-04 | 陆华 | Cell immunofluorescent staining method for differentiation induction process of bone marrow stromal cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003043486A2 (en) * | 2001-11-16 | 2003-05-30 | Tufts University | Matrix for the production of tissue engineered ligaments, tendons and other tissue |
| WO2010101883A2 (en) * | 2009-03-06 | 2010-09-10 | Mayo Foundation For Medical Education And Research | Tendon or ligament tissue engineering |
| CN102533646A (en) * | 2011-12-27 | 2012-07-04 | 陆华 | Cell immunofluorescent staining method for differentiation induction process of bone marrow stromal cells |
Non-Patent Citations (3)
| Title |
|---|
| cacy of female bone marrow-derived mesenchymal stem cells on pulmonary hypertension.《Cardiovascular Research》.2013,第100卷19-27. * |
| Rubin Tan et al..GAPDH is critical for superior effi * |
| 肺动脉高压细胞治疗研究中骨髓间充质干细胞性别对疗效的影响及其机制;谭如彬;《中国博士学位论文全文数据库医药卫生辑》;20140215;第22-24、49、55、61-63、65页 * |
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