CN117802194A - Method for detecting apoptosis resistance of mesenchymal stem cells - Google Patents
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Abstract
The invention provides an in vitro detection method for the anti-apoptosis capability of mesenchymal stem cells, which comprises the step of co-culturing the mesenchymal stem cells with vascular endothelial cells and evaluating the anti-apoptosis capability of the mesenchymal stem cells. The method can be used as a quality evaluation method of stem cells, can be used for screening stem cell products for treating ischemic diseases, and can also be used for quality release detection of cell treatment products.
Description
Cross Reference to Related Applications
The present patent application claims the priority benefit of the chinese patent application of invention application No. cn2022116767708. X filed at month 26 of 2022, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting anti-apoptosis capability of mesenchymal stem cells.
Background
Mesenchymal stem cells are widely present in various tissues, have multipotent differentiation, and are capable of differentiating into osteoblasts, chondroblasts, myocytes, neural cells, adipocytes, and the like. In recent years, the clinical value of the mesenchymal stem cells is becoming more and more important, and the clinical tests carried out on the mesenchymal stem cells prove the curative effect of the mesenchymal stem cells in various indications.
There is growing evidence that the pharmacological mechanisms of mesenchymal stem cells in the treatment of various diseases are not related to their ability to differentiate, but rather rely more on their ability to promote repair. The mesenchymal stem cell repair promoting effect comprises the aspects of inhibiting excessive inflammatory reaction in the microenvironment of damaged tissues, inhibiting apoptosis of functional cells, inhibiting fibrosis, mobilizing in-vivo stem cells to participate in repair, promoting regeneration of the functional cells and the like. In recent years, research and discovery of mesenchymal stem cells have good effect in treating cerebral infarction by taking ischemic stroke as an example, and results of a plurality of clinical experiments and animal experiments show that one of pharmacological mechanisms of treating ischemic stroke by the mesenchymal stem cells plays a role in repairing vascular injury after ischemia. Further research shows that the mesenchymal stem cells can promote the proliferation of vascular endothelial cells by secreting various nutritional factors and growth factors on one hand, and can play a role by inhibiting the expression of apoptosis-related proteins such as Caspase family, bax and the like on the other hand.
The anti-apoptosis capability of the mesenchymal stem cells is an important component of the pharmacological mechanism, and no simple, convenient and clear standard method for quantitatively evaluating the anti-apoptosis capability of the mesenchymal stem cells exists at present. In the international and domestic quality standards and technical guidelines such as the minimum requirements of mesenchymal stem cells published in 2006 of the international cell therapy society, and the standards of the human mesenchymal stem cell (T/CSCB 003) group published in 2021 of the stem cell division of the China cell biology society, the evaluation of the therapeutic efficacy of mesenchymal stem cell products is focused on the aspect of inflammation inhibition. No method for detecting the anti-apoptosis capability of the mesenchymal stem cells exists in pharmacopoeia, various standards and guidelines of various countries. In addition, the mesenchymal stem cells processed by different tissue sources, different donor sources and different processes have great differences in properties, and possibly great differences in anti-apoptosis capability, so that methods for evaluating the anti-apoptosis capability are urgently needed for quality release, indication pharmacological mechanism interpretation and curative effect prediction.
Disclosure of Invention
In order to solve the technical problems, the invention aims to establish an in vitro detection method for the anti-apoptosis capability of mesenchymal stem cells, which can be used as an in vitro evaluation method for the quality of stem cells.
The technical scheme of the invention is as follows.
In one aspect, the present invention provides an in vitro detection method for anti-apoptotic capacity of mesenchymal stem cells, comprising the steps of:
(1) Setting a control group and a co-culture group, wherein vascular endothelial cells are inoculated in the lower layer of a Transwell cell embedding vessel in the control group, culturing for 24 hours, and vascular endothelial cells and mesenchymal stem cells are respectively inoculated in the lower layer and the upper layer of the Transwell cell embedding vessel in the co-culture group, and culturing for 24 hours;
(2) Placing the lower layer and the upper layer of the Transwell cell embedding dishes of the control group and the co-culture group together to co-culture the vascular endothelial cells in the co-culture group and the mesenchymal stem cells for 24-96 hours, and continuously culturing the vascular endothelial cells in the control group for 24-96 hours;
(3) The remaining vascular endothelial cells of the co-cultured group and the control group are counted, or the apoptosis inhibition rate of the co-cultured group relative to the control group is calculated, thereby detecting the anti-apoptotic capacity of the mesenchymal stem cells.
In the detection method of the present invention, the mesenchymal stem cells may be bone marrow, umbilical cord, fat, dental pulp or placenta tissue-derived mesenchymal stem cells, or mesenchymal stem cells.
Preferably, the vascular endothelial cells are Human Umbilical Vein Endothelial Cells (HUVEC) or Human Umbilical Artery Endothelial Cells (HUAEC).
Preferably, in the step (1), the ratio of the number of cells of the inoculated mesenchymal stem cells to the number of cells of the vascular endothelial cells is 1 to 5:1, preferably 3 to 5:1, more preferably 3:1. according to a specific embodiment of the invention, the vascular endothelial cells use dedicated medium EGM and the mesenchymal stem cells use stem cell medium dmem+15% serum+growth factor. According to a specific embodiment of the present invention, the number of cells of the inoculated mesenchymal stem cells is 3×10 5 The number of vascular endothelial cells was 1X 10 5 And each.
Preferably, in step (2), the cells in the co-culture group and the control group are cultured in a co-culture medium without cytokines but containing 1 to 10% serum; preferably, the serum concentration in the medium is 1% -5%. According to a specific embodiment of the invention, the co-culture medium is 49.5% dmem+49.5% ecm+1% serum. Preferably, in step (2), the cells in the co-culture group and the control group are cultured under 1% -20% oxygen; preferably, the culture is performed under 1% to 5%, more preferably 1% oxygen. Preferably, in step (2), the cells in the co-culture group and the control group are cultured for 48 to 72 hours, preferably 72 hours. According to an embodiment of the invention, in step (2), the volume of the upper medium (free and containing mesenchymal stem cells, respectively) of the Transwell cell-embedded dishes of the control group and the co-culture group is 0.5mL; while the volume of the lower medium (containing vascular endothelial cells) was 2mL.
Preferably, in the step (3), when the number of the residual vascular endothelial cells in the co-culture group is larger than that of the residual vascular endothelial cells in the control group, or when the apoptosis inhibition rate of the co-culture group relative to the control group is not less than 20%, preferably not less than 30%, the mesenchymal stem cells are determined to have a remarkable anti-apoptosis capability. Wherein the apoptosis inhibition rate is calculated as follows: apoptosis inhibition rate= (apoptosis rate of control group-apoptosis rate of co-culture group)/apoptosis rate of control group×100%
In the context of the present invention, unless otherwise indicated, the rate of apoptosis is the early rate of apoptosis detected using flow cytometry.
According to an embodiment of the present invention, the present invention provides an in vitro method for detecting apoptosis resistance of mesenchymal stem cells, comprising:
(1) Setting a control group and a co-culture group, wherein vascular endothelial cells are inoculated in the lower layer of a Transwell cell embedding vessel in the control group, culturing for 24 hours, and vascular endothelial cells and mesenchymal stem cells are respectively inoculated in the lower layer and the upper layer of the Transwell cell embedding vessel in the co-culture group, and culturing for 24 hours; and, the cell number ratio of the inoculated mesenchymal stem cells to the vascular endothelial cells is 3:1, a step of;
(2) Placing the lower layer and the upper layer of the Transwell cell embedding dishes of the control group and the co-culture group together to enable the vascular endothelial cells in the co-culture group and the mesenchymal stem cells to be co-cultured for 72 hours, and continuously culturing the vascular endothelial cells in the control group for 72 hours; the cells in the co-culture group and the control group are cultured in a co-culture medium which contains no cytokines but 1% -5% of serum and under 1% of oxygen, the upper culture medium of the Transwell cell embedding dish is 0.5mL, and the lower culture medium is 2mL;
(3) Counting the residual vascular endothelial cells of the co-culture group and the control group, or calculating the apoptosis inhibition rate of the co-culture group relative to the control group, thereby detecting the anti-apoptosis capacity of the mesenchymal stem cells; wherein, when the number of the residual vascular endothelial cells in the co-culture group is larger than that of the residual vascular endothelial cells in the control group, or when the apoptosis inhibition rate of the co-culture group relative to the control group is more than or equal to 20%, preferably more than or equal to 30%, the mesenchymal stem cells are determined to have remarkable anti-apoptosis capability.
The invention provides an in-vitro detection method for the anti-apoptosis capability of mesenchymal stem cells, which can be used as a quality evaluation method of stem cells by evaluating the capability of the mesenchymal stem cells in resisting human vascular endothelial cell apoptosis. The method is simple and easy to implement, has clear standard, can be used for comparing mesenchymal stem cells from different donor sources, different tissue sources and different batches, and can also be used for comparing mesenchymal stem cells obtained by inducing the transdifferentiation of multipotent stem cells and endothelial cells. The method can be used for screening stem cell products for treating ischemic diseases, and can also be used for quality release detection of cell treatment products.
Experiments show that the mesenchymal stem cells with stronger anti-apoptosis capability determined by the detection method can obviously inhibit apoptosis of other cell types.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the Apoptosis rate (Apoptosis (%)) after HUVEC co-culture with human bone marrow Mesenchymal Stem Cells (MSCs); wherein a is the result of flow cytometry detection for typical apoptosis inhibition conditions; b is the statistical result of three experiments, p <0.05, p <0.01.
FIG. 2 is an inverted microscope observation of the growth of HUVEC cells in the control and co-culture groups after 72 hours of co-culture with MSC.
FIG. 3 shows apoptosis rate after HUAEC co-culture with MSC; wherein a is the result of flow cytometry detection for typical apoptosis inhibition conditions; b is the statistical result of three experiments, p <0.05.
Fig. 4 is an inverted microscope observation of the growth of control and co-cultured HUAEC cells after 72 hours of co-culture with MSCs.
FIG. 5 shows the apoptosis rate of HUVEC after various times of coculture with human bone marrow MSC.
FIG. 6 shows the apoptosis rate after HUVEC and human bone marrow MSC were co-cultured at different rates.
FIG. 7 shows HUAEC and human bone marrow MSC at different O 2 Apoptosis rate after co-culture at concentration.
FIGS. 8 and 9 show the apoptosis rate of HUVECs after co-culturing with human bone marrow MSC in the upper layer of a Transwell-embedded dish when they were in different medium volumes. In FIG. 9, the Transwell insert dish upper medium in Blank was 1mL.
FIG. 10 shows the Apoptosis rate (Apoptosis (%)) after HUVEC co-culture with different MSCs of different tissue origin.
FIG. 11 shows the apoptosis rate after neutrophils were co-cultured with different MSCs of different tissue origin.
Fig. 12 shows the apoptosis rate after neurons were co-cultured with different MSCs of different tissue origin.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified.
Example 1
Step (1): control and co-culture groups were set, 2 duplicate wells were made per group. HUVEC cells in the co-cultured group at 1X 10 5 Inoculating to the lower layer of a six-hole Transwell embedded dish (the Transwell aperture is 0.4 mu m), and the inoculating system is 2mL; human bone marrow mesenchymal stem cells at 3×10 5 Inoculated into the upper layer of a six-hole plate embedded dish, and the inoculation system is 1mL. In the control group, HUVEC cells were used at 1X 10 5 Inoculated into the lower layer of a six-well plate embedded dish, the inoculation system is 2mL, and only 1mL of culture medium is added into the upper layer. Wherein HUVEC cells use dedicated medium EGM and stem cells use stem cell medium (DMEM+15% serum+growth factor).
Step (2): after 24 hours co-cultivation was performed and the low nutrient co-culture medium (49.5% dmem+49.5% ecm+1% serum) was changed. Adding an embedding dish and a co-culture medium into the upper layer of the control group; experimental group lower HUVEC cells and upper stem cellsPut together. All groups are unified as: the volume of the culture medium was 2mL in the lower system and 0.5mL in the upper system. 37 ℃ and 5% CO 2 、1％ O 2 After culturing in the incubator for 72 hours, the cells were collected. Cells were washed 2 times with PBS, resuspended in 1× buffer (10× Annexin V binding buffer diluted 1×) with distilled water, and then counted. Taking 1×10 5 Into the U-shaped 96-well plate, the system was 100. Mu.L. Annexin V and PI were added 1. Mu.L each, mixed well and incubated at room temperature in the dark for 15min. Each well was supplemented with 100. Mu.L of a 1 Xbuffer solution dedicated to the kit (Annexin V-/PI double-stained apoptosis detection kit), mixed well and checked on a machine. The experiment was repeated three times and the results were analyzed statistically.
Step (3): the method for calculating the apoptosis inhibition rate comprises the following steps: apoptosis inhibition rate= (apoptosis rate of control group-apoptosis rate of co-culture group)/apoptosis rate of control group x 100%. The experimental results are shown in fig. 1 and 2. The apoptosis rate (early stage) of the control group was 30.18%, the apoptosis rate (early stage) of the co-culture group was 6.72%, and the apoptosis inhibition rate of the human bone marrow mesenchymal stem cells tested was 77.73%.
Example 2
Step (1): control and co-culture groups were set, 2 duplicate wells were made per group. HUAEC cells in the Co-culture group at 1X 10 5 Inoculating to the lower layer of a six-hole Transwell embedded dish (the Transwell aperture is 0.4 mu m), and the inoculating system is 2mL; human bone marrow mesenchymal stem cells at 3×10 5 Inoculated into the upper layer of a six-hole plate embedded dish, and the inoculation system is 1mL. In the control group, HUAEC cells were used at 1X 10 5 Inoculated into the lower layer of a six-well plate embedded dish, the inoculation system is 2mL, and only 1mL of culture medium is added into the upper layer. Wherein HUAEC cells use special medium EGM, and stem cells use stem cell medium (DMEM+15% serum+growth factor).
Step (2): after 24 hours co-cultivation was performed and the low nutrient co-culture medium (49.5% dmem+49.5% ecm+1% serum) was changed. Adding an embedding dish and a co-culture medium into the upper layer of the control group; the experimental group placed the lower HUAEC cells and the upper stem cells together. All groups are unified as: the volume of the culture medium was 2mL in the lower system and 0.5mL in the upper system. 37 ℃ and 5% CO 2 、1％ O 2 After culturing in the incubator for 72 hours, the cells were collected. Cells were washed 2 times with PBS, resuspended in 1× buffer (10× Annexin V binding buffer diluted 1×) with distilled water, and then counted. Taking 1×10 5 Into the U-shaped 96-well plate, the system was 100. Mu.L. Annexin V and PI were added 1. Mu.L each, mixed well and incubated at room temperature in the dark for 15min. Each well is supplemented with 100 mu L of buffer solution special for the kit, and the mixture is uniformly mixed and is detected by a machine. The experiment was repeated three times and the results were analyzed statistically.
Step (3): the method for calculating the apoptosis inhibition rate comprises the following steps: apoptosis inhibition rate= (apoptosis rate of control group-apoptosis rate of co-culture group)/apoptosis rate of control group x 100%. The experimental results are shown in fig. 3 and 4. The apoptosis rate (early stage) of the control group was 32.18%, the apoptosis rate (early stage) of the co-culture group was 21.16%, and the apoptosis inhibition rate of the human bone marrow mesenchymal stem cells tested was determined to be 34.24%.
Example 3
Bone marrow MSCs were co-cultured with HUVECs and tested for their apoptosis inhibiting ability. The experimental procedure is described with reference to example 1, with the difference that in step (2) only 20% O 2 、5％ CO 2 Co-cultivation was performed for 24 hours, 48 hours, and 72 hours, respectively.
The results are shown in Table 1 and FIG. 5.
TABLE 1 detection of different days of Co-cultivation
Note that: BLANK is control group.
The results show that the control HUVEC cells themselves have lighter apoptosis when co-cultured for 24 or 48 hours, and the apoptosis inhibition capacity (expressed as apoptosis inhibition rate) of the detected stem cells cannot be accurately judged.
Example 4
Co-mixing bone marrow MSC with HUVECCulturing, and detecting apoptosis inhibition ability of MSC. The experimental procedure is described with reference to example 1, with the only difference that the ratio of the number of two cells in step (1) is 1:1, 1:2, 1:3 or 1:5, respectively, in step (2) at 20% O 2 、5％ CO 2 Co-cultivation was performed for 3 days.
The results are shown in Table 2 and FIG. 6.
TABLE 2 detection of the quantitative ratios of the different Co-cultured cells
| Group of | Apoptosis rate (%) | Apoptosis inhibition rate (%) |
| BLANK | 43.64 | / |
| 1:1MSC | 35.73 | 18.13 |
| 1:2MSC | 38.76 | 11.18 |
| 1:3MSC | 26.78 | 38.63 |
| 1:5MSC | 30.68 | 29.70 |
Note that: BLANK is control group.
The results show that when the ratio of the two cell numbers is 1:1 (1X 10) 5 :1×10 5 ) Or 1:2 (1X 10) 5 :2×10 5 ) In the case of the HUVEC cells in the control group and the co-culture group, the apoptosis degree is close to that of the HUVEC cells in the control group and the co-culture group; and when the two cell numbers differ more than they differ, the apoptosis-inhibiting ability (expressed as apoptosis-inhibiting rate) of the detected stem cells can be judged more accurately.
Example 5
Bone marrow MSCs were co-cultured with HUAEC and tested for their apoptosis-inhibiting ability. The experimental procedure is described with reference to example 2, with the difference that O in step (2) is only 2 The concentrations were set to 20%, 5% and 1%, respectively.
The results are shown in Table 3 and FIG. 7.
TABLE 3 detection at different oxygen concentrations
Note that: BLANK is control group.
The result shows that when the oxygen concentration is 20%, the apoptosis degree of HUAEC cells of the control group and the co-culture group is close to that of HUAEC cells of the control group and the co-culture group; and when the oxygen concentration is 5% or 1%, the apoptosis inhibition ability (expressed as apoptosis inhibition rate) of the detected stem cells can be more accurately judged.
Example 6
Bone marrow MSCs were co-cultured with HUVECs and tested for their apoptosis inhibiting ability. The experimental procedure is described with reference to example 1, except that the medium in step (2) is 0.5mL or 1mL.
The results are shown in Table 4, FIG. 8 and FIG. 9.
TABLE 4 detection of different upper media
Note that: BLANK is control group.
The results show that the apoptosis difference of HUVECs in the control group and the co-culture group is larger and the apoptosis inhibition capacity (expressed as apoptosis inhibition rate) of stem cells can be accurately detected by arranging 0.5mL of culture medium on the upper layer of the Transwell embedding dish.
Example 7
Mesenchymal stem cells of different donor sources and different tissue sources were screened according to the method in example 1, from which stem cells that were effective in inhibiting HUVEC apoptosis were screened. MSC6 is mesenchymal stem cells derived from umbilical cord, and MSC1, MSC2, MSC3, MSC4, MSC5 and MSC7 are bone marrow mesenchymal stem cells derived from different donors. The results are shown in fig. 10, and the apoptosis inhibition rate of each stem cell is calculated as follows: MSC6:59.09%; MSC1:52.32%; MSC2:43.89%; MSC3:67.89%; MSC4:35.25%; MSC5:73.36%; MSC7:10.52%.
The apoptosis inhibition rate was set to be greater than 30% as effective MSC.
Example 8
The mesenchymal stem cells screened in example 7 were co-cultured with Neutrophils (NEUT) to verify their anti-apoptotic capacity. The experimental procedure is described with reference to example 1, except that the apoptosis rate is detected after 24 hours of co-culture, and the experimental results are statistically analyzed.
The results are shown in FIG. 11. The result shows that MSC with relatively strong apoptosis capability detected by the method can obviously inhibit the apoptosis of neutrophils; whereas when the measured apoptosis inhibition rate was 10.52% of that of MSC7 in co-culture with neutrophils, apoptosis of neutrophils was hardly inhibited.
Example 9
The anti-apoptotic capacity of the mesenchymal stem cells screened in example 7 was further verified using neurons (Neuron).
MSC were seeded into six well plates and co-cultured with differentiated neurons (neurons). OGD (Oxygen-Glucose De) of neurons and Co-cultured MSCs simultaneouslyPrimation, oxygen and glucose deprivation), i.e. co-culture medium using neuronal growth medium without glucose (sugarless DMEM+growth factor), and placing the cells into 1% O 2 Culturing in an incubator for 24 hours.
After 24 hours, neurons in the cell plates were digested, stained according to the procedure described in the apoptosis detection kit, and the ratio of apoptosis was detected using a flow cytometer after staining was completed.
The results are shown in FIG. 12. The result shows that MSC with relatively strong apoptosis capability detected by the method can obviously inhibit the apoptosis of neurons.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.
Claims (10)
1. An in vitro assay for the anti-apoptotic capacity of mesenchymal stem cells, said in vitro assay comprising the steps of:
(1) Setting a control group and a co-culture group, wherein vascular endothelial cells are inoculated in the lower layer of a Transwell cell embedding vessel in the control group, culturing for 24 hours, and vascular endothelial cells and mesenchymal stem cells are respectively inoculated in the lower layer and the upper layer of the Transwell cell embedding vessel in the co-culture group, and culturing for 24 hours;
(2) Placing the lower layer and the upper layer of the Transwell cell embedding dishes of the control group and the co-culture group together to co-culture the vascular endothelial cells in the co-culture group and the mesenchymal stem cells for 24-96 hours, and continuously culturing the vascular endothelial cells in the control group for 24-96 hours;
(3) The remaining vascular endothelial cells of the co-cultured group and the control group are counted, or the apoptosis inhibition rate of the co-cultured group relative to the control group is calculated, thereby detecting the anti-apoptotic capacity of the mesenchymal stem cells.
2. The method of claim 1, wherein the mesenchymal stem cells are bone marrow, umbilical cord, fat, dental pulp or placenta-derived mesenchymal stem cells.
3. The method of claim 1 or 2, wherein the vascular endothelial cells are human umbilical vein endothelial cells or human umbilical artery endothelial cells.
4. A detection method according to any one of claims 1 to 3, wherein in step (1), the ratio of the number of cells of the mesenchymal stem cells to the number of cells of the vascular endothelial cells inoculated is 1 to 5:1, preferably 3 to 5:1, more preferably 3:1.
5. the method according to any one of claims 1 to 4, wherein in step (2), the cells in the co-culture group and the control group are cultured in a co-culture medium containing 1% to 10% serum without cytokines;
preferably, the serum concentration in the medium is 1% to 5%, more preferably 1%.
6. The method according to any one of claims 1 to 5, wherein in step (2), the cells in the co-culture group and the control group are cultured under 1% to 20% oxygen; preferably, the culture is performed under 1% to 5%, more preferably 1% oxygen.
7. The method according to any one of claims 1 to 6, wherein in step (2), the cells in the co-culture group and the control group are cultured for 48 to 72 hours, preferably 72 hours;
preferably, the volume of the upper medium of the Transwell cell insert dishes of the control group and the co-culture group is 0.5mL; while the volume of the lower medium was 2mL.
8. The method according to any one of claims 1 to 7, wherein in step (3), the mesenchymal stem cells are determined to have a significant anti-apoptotic capacity when the number of vascular endothelial cells remaining in the co-cultured group is greater than the number of vascular endothelial cells remaining in the control group, or when the apoptosis inhibition rate of the co-cultured group relative to the control group is not less than 20%, preferably not less than 30%; wherein the apoptosis inhibition rate is calculated as follows:
apoptosis inhibition rate= (apoptosis rate of control group-apoptosis rate of co-culture group)/apoptosis rate of control group x 100%.
9. The method of claim 8, wherein the apoptosis rate is an early apoptosis rate detected by flow cytometry.
10. The detection method according to any one of claims 1 to 9, characterized in that the method comprises:
(1) Setting a control group and a co-culture group, wherein vascular endothelial cells are inoculated in the lower layer of a Transwell cell embedding vessel in the control group, culturing for 24 hours, and vascular endothelial cells and mesenchymal stem cells are respectively inoculated in the lower layer and the upper layer of the Transwell cell embedding vessel in the co-culture group, and culturing for 24 hours; and, the cell number ratio of the inoculated mesenchymal stem cells to the vascular endothelial cells is 3:1, a step of;
(2) Placing the lower layer and the upper layer of the Transwell cell embedding dishes of the control group and the co-culture group together to enable the vascular endothelial cells in the co-culture group and the mesenchymal stem cells to be co-cultured for 72 hours, and continuously culturing the vascular endothelial cells in the control group for 72 hours; the cells in the co-culture group and the control group are cultured in a co-culture medium which contains no cytokines but 1% -5% of serum and under 1% of oxygen, the upper culture medium of the Transwell cell embedding dish is 0.5mL, and the lower culture medium is 2mL;
(3) Counting the residual vascular endothelial cells of the co-culture group and the control group, or calculating the apoptosis inhibition rate of the co-culture group relative to the control group, thereby detecting the anti-apoptosis capacity of the mesenchymal stem cells; wherein, when the number of the residual vascular endothelial cells in the co-culture group is larger than that of the residual vascular endothelial cells in the control group, or when the apoptosis inhibition rate of the co-culture group relative to the control group is more than or equal to 20%, preferably more than or equal to 30%, the mesenchymal stem cells are determined to have remarkable anti-apoptosis capability.
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