Application of CD200 in evaluation of biological functions of mesenchymal stem cells

Abstract

The invention relates to the technical field of biology, in particular to application of CD200 in evaluation of biological functions of mesenchymal stem cells. The biological efficacy of the cells can be rapidly judged by detecting the positive rate and the average fluorescence intensity of the CD200 on the surfaces of the mesenchymal stem cells through flow cytometry, and the method is simple, convenient and rapid, has a wide application prospect and is worthy of popularization and application. According to the invention, the expression level of the CD200 on the surface of the mesenchymal stem cell and the biologic function (secretion level of anti-inflammatory factors PGE2 and IDO, and inhibition rate on TNF-alpha and IFN-gamma secreted by PBMC) of the CD200 are illuminated for the first time, and the biological function of the umbilical cord mesenchymal stem cell is evaluated by adopting a method for detecting the positive rate of the CD200 and MFI for the first time. When the CD200 positive rate is greater than 40% and the average fluorescence intensity MFI is greater than 500, the TNF alpha secretion inhibition rate is greater than 40%, and the IFN gamma secretion inhibition rate is greater than 20%.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of CD200 in evaluating the biological functions of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) originate from the mesoderm and are a type of adult stem cell with self-replication, high self-renewal capacity, multidirectional differentiation potential, and immune regulation capabilities after continuous subculture. The main functions of MSCs include immune regulation, angiogenesis, and tissue repair. Their immune regulation is mainly achieved through paracrine and direct contact. The mechanism of action of MSCs in mediating immune regulation is very complex and difficult to determine the standard, making the evaluation of their biological efficacy a difficult problem for their quality control.

[0003] Currently, the quality evaluation method for mesenchymal stem cell drugs mainly involves detecting their inhibition on the proliferation of activated peripheral blood mononuclear cells and the inhibition rate of inflammatory factors TNFα\IFNγ secretion, as well as the detection of specific lymphocyte subsets. The operation is complex and costly. Summary of the Invention

[0004] In light of this, the present invention aims to provide a method for evaluating the biological efficacy of mesenchymal stem cells using CD200. This method differs from conventional methods of assessing the biological efficacy of mesenchymal stem cells by detecting cytokines or proteins using ELISA. This method rapidly determines the biological efficacy of mesenchymal stem cells by detecting the positive rate and mean fluorescence intensity of CD200 on the surface of mesenchymal stem cells using flow cytometry. This method is simple, rapid, and has broad application prospects, making it worthy of widespread application.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides the use of CD200 in evaluating the biological function of mesenchymal stem cells.

[0007] In some specific embodiments of the present invention, the biological function includes any of the following:

[0008] (I) secretion level of anti-inflammatory factors;

[0009] (II) Inhibition rate of PBMC secretion of pro-inflammatory factors.

[0010] In some specific embodiments of the present invention, the anti-inflammatory factors include one or more of PGE2 or IDO;

[0011] The pro-inflammatory factors include one or more of TNF-α and IFN-γ.

[0012] In some specific embodiments of the present invention, the mesenchymal stem cells include one or more of umbilical cord, bone marrow, placenta or adipose-derived mesenchymal stem cells;

[0013] Preferably, the mesenchymal stem cells include umbilical cord-derived mesenchymal stem cells.

[0014] In some specific embodiments of the present invention, the positive rate and / or mean fluorescence intensity of CD200 is positively correlated with the biological function of mesenchymal stem cells.

[0015] In some specific embodiments of the present invention, when the positive rate of CD200 is greater than 40% and the mean fluorescence intensity (MFI) of CD200 is greater than 500, the TNFα secretion inhibition rate is greater than 40%, and the IFNγ secretion inhibition rate is greater than 20%.

[0016] In a second aspect, the present invention further provides a method for detecting the biological function of mesenchymal stem cells, comprising the following steps:

[0017] Mesenchymal stem cells were cultured in defined medium, serum-containing (FBS) medium, or platelet lysate-containing medium, and the supernatant was collected;

[0018] Flow cytometry was used to detect the positive rate and / or mean fluorescence intensity (MFI) of CD200;

[0019] The positive rate and / or mean fluorescence intensity (MFI) of CD200 is positively correlated with the biological function of the mesenchymal stem cells.

[0020] In some specific embodiments of the present invention, the biological function includes any of the following:

[0021] (I) secretion level of anti-inflammatory factors;

[0022] (II) Inhibition rate of PBMC secretion of pro-inflammatory factors.

[0023] In some specific embodiments of the present invention, the anti-inflammatory factors include one or more of PGE2 or IDO;

[0024] The pro-inflammatory factors include one or more of TNF-α or IFN-γ;

[0025] The mesenchymal stem cells include mesenchymal stem cells derived from umbilical cord, bone marrow, placenta or fat;

[0026] Preferably, the mesenchymal stem cells include umbilical cord-derived mesenchymal stem cells.

[0027] In some specific embodiments of the present invention, the positive rate and / or mean fluorescence intensity of CD200 is positively correlated with the biological function of mesenchymal stem cells;

[0028] When the positive rate of CD200 is greater than 40% and the mean fluorescence intensity (MFI) of CD200 is greater than 500, the TNFα secretion inhibition rate is greater than 40%, and the IFNγ secretion inhibition rate is greater than 20%.

[0029] The present invention provides surface markers reflecting the functional status of umbilical cord mesenchymal stem cells, which can be used for detection using flow cytometry in clinical and basic scientific research, to accurately classify the functional status of stem cells, clarify their targets and molecular mechanisms as stem cell efficacy molecules, improve the quality evaluation system, and enhance the clinical treatment effect of stem cells.

[0030] The present invention can quickly determine the biological efficacy of mesenchymal stem cells by detecting the positive rate and mean fluorescence intensity of CD200 on the surface of mesenchymal stem cells by flow cytometry. This method is simple and fast, has broad application prospects, and is worthy of promotion and application. The present invention is the first to clarify that the expression level of CD200 on the surface of mesenchymal stem cells is significantly positively correlated with their biological functions (secretion levels of anti-inflammatory factors PGE2 and IDO, and inhibition rate of PBMC secretion of TNF-α and IFN-γ) within a certain range, and is the first to use flow cytometry to detect the CD200 positive rate and MFI to evaluate the biological function of umbilical cord mesenchymal stem cells. When the CD200 positive rate is greater than 40% and the mean fluorescence intensity MFI is greater than 500, the TNFα secretion inhibition rate is greater than 40% and the IFNγ secretion inhibition rate is greater than 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0032] Figure 1 Schematic diagram showing the analysis of CD200 positivity and mean fluorescence intensity of umbilical cord mesenchymal stem cells under different batches and passages; a: CD200 positivity detection; b: Correlation analysis between CD200 positivity and mean fluorescence intensity;

[0033] Figure 2 Schematic diagram showing the analysis of CD200 positive rate and mean fluorescence intensity of umbilical cord mesenchymal stem cells under different culture conditions; a: CD200 positive rate detection; b: Correlation analysis between CD200 positive rate and mean fluorescence intensity;

[0034] Figure 3Schematic diagram showing the correlation analysis between the secretion of the anti-inflammatory factor PGE2 and the expression of CD200 in multiple batches of umbilical cord mesenchymal stem cells; wherein, a: PGE2 secretion detection; b: correlation analysis between the mean fluorescence intensity of CD200 and PGE2 secretion;

[0035] Figure 4 Schematic diagram showing the correlation analysis between the secretion of the anti-inflammatory factor IDO and the expression of CD200 in multiple batches of umbilical cord mesenchymal stem cells; a: IDO secretion detection; b: Correlation analysis between the mean fluorescence intensity of CD200 and IDO secretion;

[0036] Figure 5 Schematic diagram showing the correlation analysis between the inhibition rate of pro-inflammatory factor secretion and CD200 expression of multiple batches of umbilical cord mesenchymal stem cells; among them, a: TNFα secretion inhibition rate detection; b: correlation analysis between CD200 positivity rate and TNFα secretion inhibition rate; c: IFNγ secretion inhibition rate detection; d: correlation analysis between CD200 mean fluorescence intensity and IFNγ secretion inhibition rate; e: CD200 positivity rate detection; f: CD200 mean fluorescence intensity detection. DETAILED DESCRIPTION

[0037] The present invention discloses the application of CD200 in evaluating the biological functions of mesenchymal stem cells. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0038] CD200 is a highly glycosylated transmembrane protein and a member of the immunoglobulin superfamily. It is widely expressed on the surfaces of T cells, B cells, vascular endothelial cells, APCs, and MSCs. Studies have shown that CD200 cell surface expression varies significantly among mesenchymal stem cells derived from different tissues, with umbilical cord mesenchymal stem cells expressing the highest proportion of CD200. Under hypoxic or inflammatory conditions, CD200 expression is upregulated on MSCs, suggesting that it may be involved in the regulation of inflammatory processes in MSCs.

[0039] Based on the current phenotypic research on CD200 in mesenchymal stem cells and the lack of relevant reports on the use of this surface molecule to evaluate the biological efficacy of mesenchymal stem cells, the expression of CD200 under existing process conditions and its relationship with anti-inflammatory factors (such as IDO and PGE2 secretion levels) and pro-inflammatory factors (such as TNFα and IFNγ secretion inhibition rate) were explored. CD200 was used to evaluate the biological efficacy of mesenchymal stem cells, and the positive rate and mean fluorescence intensity (MFI) of CD200 were analyzed by flow cytometry to evaluate the biological efficacy of mesenchymal stem cells. The high and low MFI represent the high and low abundance of target antigen expression. Flow cytometry detection is more convenient than conventional ELISA detection.

[0040] The present invention provides a method for evaluating the biological efficacy of mesenchymal stem cells using CD200, which uses an antibody to label the membrane protein CD200 on the surface of mesenchymal stem cells, and uses a flow cytometer to detect the positive rate and mean fluorescence intensity (MFI) of the cells CD200. The biological efficacy of the mesenchymal stem cells is evaluated by detecting the expression level of CD200 on the surface of the mesenchymal stem cells. The biological efficacy refers to the expression of anti-inflammatory factors PGE2, IDO (such as Figure 2 、 Figure 3 The secretion level of TNF-α and IFN-γ of PBMC is shown in Figure 2. The mesenchymal stem cells are derived from umbilical cord, bone marrow, placenta or adipose tissue.

[0041] The present invention provides a method for evaluating the biological efficacy of mesenchymal stem cells using CD200. The specific evaluation method is as follows:

[0042] 1. Compared with the traditional experimental method including collecting stem cell supernatant and detecting cytokines by ELISA method, the present invention directly uses stem cell mild digestion enzyme to digest and collect cell marker CD200 antibody for incubation, and performs flow cytometry to obtain the positive rate and mean fluorescence intensity MFI of CD200. The present invention omits the complicated steps of collecting culture supernatant, packaging and freezing and then thawing, ELISA detection, etc., avoids the problem of easy degradation due to unstable cytokines, and the operation steps are simpler and faster, saving time and labor, and being more economical and efficient in time. At the same time, it avoids the possible problem of human contamination, and the membrane protein index is more stable than the cytokine index, not easy to degrade, and easy to detect. This method can be applied to the master cell bank, working cell bank and finished product stage, the centralized inspection of multiple batches of samples, the direct detection of sampled samples, and the test results can represent the true situation of the sample.

[0043] 2. The present invention illustrates that the expression level of CD200 on the surface of mesenchymal stem cells is significantly positively correlated with their biological efficacy level (secretion levels of anti-inflammatory factors PGE2 and IDO, and inhibition rate of PBMC secretion of TNF-α and IFN-γ) within a certain range, and for the first time uses flow cytometry to detect CD200 positivity rate and MFI to evaluate the biological efficacy of umbilical cord mesenchymal stem cells.

[0044] 3.CD200 is expressed under different culture conditions including serum-containing (FBS) medium, serum-free (FBS) medium and defined medium.

[0045] The raw materials and reagents used in the application of CD200 provided by the present invention in evaluating the biological functions of mesenchymal stem cells can all be purchased from the market.

[0046] The present invention will be further described below in conjunction with the embodiments:

[0047] Example 1

[0048] The positive rate and mean fluorescence intensity of CD200 on the surface of umbilical cord mesenchymal stem cells from different people were detected. The specific implementation steps are as follows:

[0049] Flow cytometry was used to detect the expression levels of CD200 on the surface of mesenchymal stem cells in multiple samples:

[0050] (1) Take mesenchymal stem cells, discard the culture supernatant, wash once with physiological saline (Huarun Shuanghe, H11021192), add 4ml Tryple TM Digestion solution (Gibco, 12563-029) was added at room temperature for 2-5 minutes. 8 ml of culture medium (Gibco, 11835-030) was added to terminate the digestion. The centrifuge parameters were set to 400g and centrifuged for 8 minutes. The cells were resuspended in serum-free culture medium (Gibco, 11835-030) and counted using AOPI (Nexcelom, CS2-0106). The AOPI staining solution and cell suspension were mixed in equal proportions and added to the detection plate for detection.

[0051] Take the mesenchymal stem cells, discard the culture supernatant, wash once with normal saline (Huarun Shuanghe, H11021192), add 4ml Tryple TM Digestion solution (Gibco, 12563-029) was added at room temperature for 2-5 minutes. 8 ml of culture medium (Gibco, 11835-030) was added to terminate the digestion. The centrifuge parameters were set to 400 g and centrifuged for 8 minutes. The cells were resuspended in 3 ml of serum-free culture medium (Gibco, 11835-030). 100 μl of the cell suspension was taken for AOPI (Nexcelom, CS2-0106) counting.

[0052] According to the counting results, umbilical cord mesenchymal stem cells of different batches and passages under different serum-free culture conditions were transferred to flow cytometry tubes at a rate of (0.5-1)*106 cells / tube. Two sample tubes and two isotype control tubes were prepared for each cell type. After centrifugation at 1500rpm / 5min, the cell pellet was retained. After washing twice with physiological saline, 100μl was resuspended and APC-IgG isotype control antibody (Biolgend) and APC-CD200 antibody (Biolgend) were added and vortexed to mix. The cells were incubated in the dark at 4°C for 30min, and the cells were washed with physiological saline. The cells were centrifuged at 1500rpm for 5min and washed twice to remove non-specific binding. The cells were resuspended in 400μl of physiological saline. The positive rate of CD200 was detected by flow cytometry within 1hur and the MFI and the correlation between the two were calculated. The results are shown in the figure. Figure 1 As shown. Figure 1 a It can be seen that there are significant individual differences in the positive rate and MFI of CD200 of mesenchymal stem cells of different batches and passages. The average positive rate of CD200 is (48%±13.9)%, and the MFI of CD200 is 2228.8±762.1. The average positive rate of CD200 in umbilical cord mesenchymal stem cells at P4 was (53% ± 15.7)%, and the MFI of CD200 was 1950.8 ± 731.2. The average positive rates of CD200 in umbilical cord mesenchymal stem cells at P5 and P7 were (48.2% ± 11.0)% and (44.9% ± 16.2)%, respectively, and the MFI of CD200 was 2660.0 ± 864.7 and 2099.8 ± 813.3. The positive rate of CD200 showed a downward trend with the increase of umbilical cord mesenchymal stem cell generations. Spearman correlation analysis (1b) of the positive rate and MFI of cell surface CD200 showed a significant positive correlation between the two (r 2 =0.9217,p<0.0001).

[0053] (2) Umbilical cord mesenchymal stem cells were collected and cultured in different conditions of complete medium (Gibco, 11835-030) containing 10% FBS (Gibco, 10091-148), serum-free medium (Gibco, 11835-030) and defined medium (Youkang Biotechnology, NC0103) for flow cytometry analysis, as described above. The results are shown in Figure 2. Figure 2 As shown by Figure 2a It can be seen that the positive rate and MFI of CD200 of umbilical cord mesenchymal stem cells under different culture medium conditions were different. The average positive rate of CD200 was (41.2% ± 25.5)%, and the MFI of CD200 was 2242.1 ± 1829.4. Spearman correlation analysis (2b) of the positive rate and MFI of cell surface CD200 showed that the two were significantly positively correlated (r 2 =0.8950,p<0.0001).

[0054] Example 2

[0055] The factor secretion function of multiple batches of umbilical cord mesenchymal stem cells was evaluated, and correlation analysis was performed based on their CD200 expression levels. The specific implementation method is as follows:

[0056] (1) Flow cytometry detection of CD200 expression levels on the surface of mesenchymal stem cells in multiple samples: the method was the same as that in Example 1.

[0057] (2) Factor secretion testing of multiple batches of stem cells:

[0058] Co-culture experiment: Take the cultured mesenchymal stem cells, discard the culture supernatant, wash once with physiological saline (Huarun Shuanghe, H11021192), add 4ml Tryple TM Digestion solution (Gibco, 12563-029) was added at room temperature for 2-5 minutes. 8 ml of culture medium (Gibco, 11835-030) was added to terminate the digestion. The centrifuge parameters were set to 400g and centrifuged for 8 minutes. The cells were resuspended in serum-free culture medium (Gibco, 11835-030) and counted using AOPI (Nexcelom, CS2-0106). The AOPI staining solution and cell suspension were mixed in equal proportions and added to the detection plate for detection.

[0059] Take the mesenchymal stem cells, discard the culture supernatant, wash once with normal saline (Huarun Shuanghe, H11021192), add 4ml Tryple TM Digestion solution (Gibco, 12563-029) was added at room temperature for 2-5 minutes. 8 ml of culture medium (Gibco, 11835-030) was added to terminate the digestion. The centrifuge parameters were set to 400 g and centrifuged for 8 minutes. The cells were resuspended in 3 ml of 1640 complete culture medium (Gibco, 11835-030). 100 μl of the cell suspension was taken for AOPI (Nexcelom, CS2-0106) counting.

[0060] Umbilical cord mesenchymal stem cells were taken and resuspended in 1640 complete medium (Gibco, 11835-030). According to the counting results, 2*105 2 ml of cells were plated on a 6-well plate. Observe under a microscope to ensure that the cells were evenly distributed on the bottom of the plate. The plate was then placed in a 5% CO2 incubator for culture. Peripheral blood mononuclear cells (PB150F, Miaoshun Biotechnology) were resuspended in 1640 complete medium (supplemented with 10 μg / ml of PHA). After 4-6 hours, when the mesenchymal stem cells adhered to the plate, 2×10 cells were plated per well. 6 The PBMC suspension was added to the MSCs at 2 ml per 100 mL of culture medium and mixed again. The control group received PHA-activated PBMCs, while the experimental group co-cultured MSCs and PHA-activated PBMCs. After 72 hours of culture, the supernatant was collected and centrifuged at 2500 rpm for 10 minutes. The cell culture supernatant was aspirated and aliquoted into 1 ml tubes and frozen at -80°C for analysis of PGE2, IDO, IFN-γ, and TNF-α levels in the supernatant.

[0061] IDO and PGE2 detection methods:

[0062] The supernatant to be tested was taken out of the -80℃ refrigerator, thawed on ice, and ELISA detection was performed using the IDO detection kit (Sangon Biotechnology, sD711266-0096) and the PGE2 detection kit (biotechne SKGE004B), as follows: ② The ELISA reagents were equilibrated at room temperature for about 20 minutes before use, and all sample wells, control wells, and standard wells were prepared in duplicate; ② 100 μL of standard and sample were added to the corresponding wells and incubated at 37℃ for 1.5 hours; ③ Wash the plate 4 times, using 350 μL of wash solution each time; ④ 100 μL of detection antibody was added to each well and incubated at 37℃ for 1 hour; ⑤ After incubation, wash the plate according to step ③; ⑥ 100 μL of color development solution was added to each well and incubated at 37℃ in the dark for 15-30 minutes; ⑦ 100 μL of stop solution was added to each well, and the color of the well plate changed from blue to yellow; ⑧ Read the OD (450 nm) value within 5 minutes.

[0063] The expression levels of human IFN-γ and TNF-α in the supernatant were detected using human IFN-γ and TNF-α ELISA kits (Sanggong).

[0064] The expression levels in the supernatant were detected using an IDO detection kit (Sanggong) and a PGE2 detection kit (Biotechne SKGE004B).

[0065] Table 1

[0066]

[0067] (3) Correlation analysis was performed on the CD200 expression levels of multiple batches of mesenchymal stem cells and the secretion levels of PGE2 and IDO. The analysis results are as follows: Figure 3 and Figure 4 shown.

[0068] After approval by the hospital ethics committee, the mother signed an informed consent form and health questionnaire to ensure that she had no infectious diseases or family hereditary diseases. The baby's umbilical cord was collected, Wharton's jelly was separated, and umbilical cord mesenchymal stem cells were isolated using the tissue block adhesion method.

[0069] Depend on Figure 3 a and Figure 4 a It can be seen that the secretion of PGE2 and IDO by mesenchymal stem cells from different donors (mesenchymal stem cells isolated by traditional tissue adhesion method) is different, among which the secretion of PGE2 is 9.6±9.0 (ng / ml) and the secretion of IDO is 5.6±0.4 (ng / ml). The expression of CD200 on the cell surface is different from that of PGE2 ( Figure 3 b) IDO Figure 4 b) Spearman correlation analysis of secretion showed that the mean fluorescence intensity of CD200 was significantly positively correlated with the secretion of PGE2 and IDO, with a correlation coefficient of r 2 They were 0.7201 and 0.4841 respectively, indicating that the higher the CD200 expression, the higher the secretion of anti-inflammatory related factors PGE2 and IDO, and the stronger the anti-inflammatory ability.

[0070] (4) The CD200 expression levels of multiple batches of mesenchymal stem cells and their effects on the secretion of IFN-γ and TNF-α by activated PBMC were detected using human IFN-γ and TNF-α ELISA kits (Sanggong), and the inhibition rate (%) was calculated according to the following formula:

[0071] IFN-γ inhibition rate (%) = [1-IFN-γ (experimental group) / IFN-γ (control group)] * 100%

[0072] TNF-α inhibition rate (%) = [1-TNF-α (experimental group) / TNF-α (control group)] * 100%

[0073] The results are as follows Figure 5 As shown. Figure 5 As shown in Figures 5a and 5c, the secretion inhibition rates of TNFα and IFN-γ of mesenchymal stem cells from different sources were different, with the secretion inhibition rate of TNFα being 73.0±10.9(%) and the secretion inhibition rate of IFN-γ being 50.6±16.4(%). Figure 5 b) IFN-γ ( Figure 5 d) Spearman correlation analysis of secretion showed that the mean fluorescence intensity of CD200 was significantly positively correlated with the secretion inhibition rate of TNFα and IFN-γ, with a correlation coefficient of r 2The values ​​were 0.6111 and 0.7421, respectively, indicating that the higher the CD200 expression, the higher the inhibition rate of pro-inflammatory cytokines secretion and the stronger the anti-inflammatory ability. When the CD200 positive rate is greater than 40% and the mean fluorescence intensity (MFI) is greater than 500, the inhibition rate of TNFα secretion is greater than 40% and the inhibition rate of IFNγ secretion is greater than 20%. ( Figure 5 a, 5c, 5e, 5f).

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of CD200 in evaluating the biological functions of mesenchymal stem cells.

2. The use according to claim 1, characterized in that The biological functions include any of the following: (I) secretion level of anti-inflammatory factors; (II) Inhibition rate of PBMC secretion of pro-inflammatory factors.

3. The use according to claim 1 or 2, characterized in that The anti-inflammatory factors include one or more of PGE2 or IDO; The pro-inflammatory factors include one or more of TNF-α and IFN-γ.

4. The use according to any one of claims 1 to 3, characterized in that The mesenchymal stem cells include one or more of umbilical cord, bone marrow, placenta or adipose-derived mesenchymal stem cells; Preferably, the mesenchymal stem cells include umbilical cord-derived mesenchymal stem cells.

5. The use according to any one of claims 1 to 4, characterized in that The positive rate and / or mean fluorescence intensity of CD200 are positively correlated with the biological function of mesenchymal stem cells.

6. The use according to any one of claims 1 to 5, characterized in that When the positive rate of CD200 is greater than 40% and the mean fluorescence intensity (MFI) of CD200 is greater than 500, the TNFα secretion inhibition rate is greater than 40%, and the IFNγ secretion inhibition rate is greater than 20%.

7. A method for detecting the biological function of mesenchymal stem cells, characterized in that: The steps include: Mesenchymal stem cells were cultured in defined medium, serum-containing (FBS) medium, or platelet lysate-containing medium, and the supernatant was collected; Flow cytometry was used to detect the positive rate and / or mean fluorescence intensity (MFI) of CD200; The positive rate and / or mean fluorescence intensity (MFI) of CD200 is positively correlated with the biological function of the mesenchymal stem cells.

8. The detection method according to claim 7, wherein The biological functions include any of the following: (I) secretion level of anti-inflammatory factors; (II) Inhibition rate of PBMC secretion of pro-inflammatory factors.

9. The detection method according to claim 7 or 8, wherein The anti-inflammatory factors include one or more of PGE2 and IDO; The pro-inflammatory factors include one or more of TNF-α or IFN-γ; The mesenchymal stem cells include mesenchymal stem cells derived from umbilical cord, bone marrow, placenta or fat; Preferably, the mesenchymal stem cells include umbilical cord-derived mesenchymal stem cells.

10. The detection method according to any one of claims 7 to 9, characterized in that: The positive rate and / or mean fluorescence intensity of CD200 are positively correlated with the biological function of mesenchymal stem cells; When the positive rate of CD200 is greater than 40% and the mean fluorescence intensity (MFI) of CD200 is greater than 500, the TNFα secretion inhibition rate is greater than 40%, and the IFNγ secretion inhibition rate is greater than 20%.

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