CN117949372A - Method for evaluating biological efficacy of mesenchymal stem cells by using CD273 - Google Patents
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Abstract
The invention relates to the field of biotechnology, in particular to a method for evaluating biological efficacy of mesenchymal stem cells by using CD 273. The invention aims to provide a method for evaluating biological efficacy of mesenchymal stem cells by using CD273, which is different from the prior method for evaluating the biological efficacy of mesenchymal stem cells by detecting cytokines or proteins through ELISA, can rapidly judge the biological efficacy of the cells by detecting the expression level of CD273 on the surface of the mesenchymal stem cells through flow cytometry, is simple, convenient and rapid, has wide application prospect, and is worthy of popularization and application.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for evaluating biological efficacy of mesenchymal stem cells by using CD 273.
Background
Mesenchymal stem cells (MESENCHYMAL STEM CELL, MSCs) are derived from mesoderm, and are an adult stem cell with self-replication, high self-renewal capacity, multi-directional differentiation potential and immune regulation capacity after continuous subculture. MSCs are involved in the regeneration of tissues such as bone, cartilage, muscle, fat, tendon, liver, etc. The immune regulation is mainly realized through factor secretion and direct contact between cells, but the specific mechanism is not clear. The main factors involved in immune regulation of stem cells are reported to include PGE2, IDO, IL-10, TSG-6, TGF-beta 1, etc., and the membrane proteins on the cell surface are also reported to be involved in immune regulation processes, such as ICAM-1, VCAM-1, galectin-1, TLR3, TLR4, PD-L1, PD-L2, etc. In view of the multiple biological functions of mesenchymal stem cells, the method for evaluating the immunoregulatory function of the mesenchymal stem cell drug mainly detects the inhibition of PBMC proliferation and TNF-alpha secretion, and the detection of specific lymphocyte subpopulations (Th 1/Treg/Th 17), and has complex operation content and high cost. In view of the high cost of batch detection of mesenchymal stem cell drugs, drug development workers have attempted to use alternative functional indicators to assess the biological efficacy of mesenchymal stem cells, i.e., to link their biological efficacy with their own expressed key cytokines or proteins, and to take this as a method of quality assessment. Alternative markers currently known for evaluating the biological efficacy of mesenchymal stem cells include PGE2, TNFRI, galectin-3, and the like. It is important to explore the action mechanism of stem cells, define biological efficacy molecules, and develop a convenient and rapid detection method to evaluate biological functions.
CD273 (B7-DC, also known as programmed cell death ligand 2, PDL 2) is a newly discovered immunomodulatory protein belonging to the B7 family, which is one of the ligands of programmed cell death protein 1 (PD 1), with an amino acid sequence homology of about 40% with another ligand subtype PD-L1. CD273 is located on chromosome 9p24.1, and in healthy humans, CD273 is mainly expressed in immune cells such as Antigen Presenting Cells (APC), B cells, and activated T cells. As a novel immune checkpoint, CD273 is highly expressed on the surface of tumor cells and is closely related to poor prognosis in tumor patients. Studies show that the CD273 can improve the tolerance of T cells after being combined with the receptor thereof, induces the inhibition effect on the activation/proliferation of the T cells, increases the transformation of T helper cells to Foxp3+ Treg cells, prevents the cell killing effect of the T cells in cancer cells, inhibits the immune system, participates in the escape of tumor cells, can be used as a target point of tumor immunotherapy, can be used as a competitive ligand of PDL1, has higher affinity with the PD1, and has a difference with PDL1 in dynamic level and expression level development. Studies have shown that under inflammatory conditions, PDL2 expression changes much more than PDL1 and enhances immune function by directly activating DCs.
Considering that the current research on CD273 in MSC is shallow, the proliferation inhibition effect on T cells is mainly focused, and the biological efficacy evaluation of the CD273 on MSC, especially on the secretion level of anti-inflammatory factors PGE2 and IDO under the condition of macrophage polarization, the function inhibition of inflammatory factors secreted by cells has not been reported. Furthermore, there is no report on the evaluation of the biological efficacy of the MSC by directly detecting the positive proportion and the average fluorescence intensity of CD273 by flow cytometry.
Disclosure of Invention
In view of this, the present invention provides methods for evaluating the biological efficacy of mesenchymal stem cells using CD 273.
The present invention provides methods for evaluating the biological efficacy of mesenchymal stem cells using CD 273. The invention has the beneficial effects that. The invention discovers a surface marker capable of reflecting the functional state of mesenchymal stem cells, so that the functional state of the stem cells is accurately divided by using flow cytometry in clinical and basic scientific researches, the functional state of the stem cells is definitely used as an action target point and a molecular mechanism of stem cell efficacy molecules, a quality evaluation system is perfected, and the clinical treatment effect of the stem cells is improved.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides application of CD273 in preparing a product for evaluating biological efficacy of mesenchymal stem cells.
In some embodiments of the invention, said assessing mesenchymal stem cell biological efficacy comprises detecting any of:
(1) Anti-inflammatory factor content; and/or
(2) Inhibition of cytokines; and/or
(3) Lymphocyte proliferation inhibition rate and/or Treg promotion rate.
In some embodiments of the invention, the anti-inflammatory agent comprises PGE2 and/or IDO;
the cytokines include TNF-alpha and/or IFN-gamma.
In some embodiments of the invention, the expression level of CD273 is significantly positively correlated with the level of biological efficacy of the mesenchymal stem cells.
In some embodiments of the invention, the criteria for the evaluation are: when the CD273 positive rate is more than 90%, the TNF-alpha inhibition rate of the mesenchymal stem cells is more than 60% and/or the IFN-gamma inhibition rate is more than 80%.
In some embodiments of the invention, the source of mesenchymal stem cells comprises one or more of umbilical cord, bone marrow, placental fat, dental pulp, or synovium.
In some embodiments of the invention, the source of the mesenchymal stem cells is umbilical cord.
The invention also provides a method for evaluating the biological efficacy of the mesenchymal stem cells by utilizing CD273, which is characterized in that the cultured mesenchymal stem cells are incubated with a CD273 antibody, and the biological efficacy of the mesenchymal stem cells is obtained by detecting the CD273 positive rate and the MFI by utilizing a flow cytometry.
In some embodiments of the invention, the incubation is: light is protected from light at 4 ℃ for 30min.
In some embodiments of the invention, said assessing mesenchymal stem cell biological efficacy comprises detecting any of:
(1) Anti-inflammatory factor content; and/or
(2) Inhibition of cytokines; and/or
(3) Lymphocyte proliferation inhibition rate and/or Treg promotion rate.
In some embodiments of the invention, the anti-inflammatory agent comprises PGE2 and/or IDO;
the cytokines include TNF-alpha and/or IFN-gamma.
In some embodiments of the invention, the criteria for the evaluation are: when the CD273 positive rate is more than 90%, the TNF-alpha inhibition rate of the mesenchymal stem cells is more than 60% and/or the IFN-gamma inhibition rate is more than 80%.
In some embodiments of the invention, the culture medium employed in the culture comprises one or more of a defined medium, an FBS-containing medium, or a platelet lysate-containing medium;
the source of the mesenchymal stem cells comprises one or more of umbilical cord, bone marrow, placental fat, dental pulp or synovium.
The present invention provides methods for evaluating the biological efficacy of mesenchymal stem cells using CD 273. The invention aims to provide a method for evaluating biological efficacy of mesenchymal stem cells by using CD273, which is different from the prior method for evaluating the biological efficacy of mesenchymal stem cells by detecting cytokines or proteins through ELISA, can rapidly judge the biological efficacy of the cells by detecting the expression level of CD273 on the surface of the mesenchymal stem cells through flow cytometry, is simple, convenient and rapid, has wide application prospect, and is worthy of popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram showing the analysis of the positive rate and average fluorescence intensity of mesenchymal stem cell CD273 under different culture conditions for a plurality of batches in example 1; wherein A is an analysis schematic diagram of the positive rate of the mesenchymal stem cells CD273 under different culture conditions of a plurality of batches; b is an analysis schematic diagram of the average fluorescence intensity of the mesenchymal stem cells CD273 under different culture conditions of a plurality of batches;
FIG. 2 is a graph showing a correlation analysis of the amount of anti-inflammatory factor secretion and CD273 expression of mesenchymal stem cells in a plurality of batches in example 2; wherein, A is a correlation analysis schematic diagram of the secretion amount of the anti-inflammatory factor PGE2 of the mesenchymal stem cells and the expression of CD 273; b is a correlation analysis schematic diagram of the secretion amount of anti-inflammatory factors IDO and the expression of CD273 of the mesenchymal stem cells in batches;
FIG. 3 is a graph showing a correlation analysis of the inhibition rate of the secretion of proinflammatory factors and the expression of CD273 in a plurality of batches of mesenchymal stem cells of example 3; a is a schematic diagram of correlation analysis of the inhibition rate of TNF-alpha secretion of mesenchymal stem cell pro-inflammatory factors and CD273 expression; b is a correlation analysis schematic diagram of the inhibition rate of IFN-gamma secretion of the batch mesenchymal stem cell pro-inflammatory factor and the expression of CD 273;
FIG. 4 is a graph showing a correlation analysis of lymphocyte inhibition rate and CD273 expression by a plurality of batches of mesenchymal stem cells in example 4;
Figure 5 shows a graphical representation of a correlation analysis of the percentage Treg promotion and CD273 expression of a plurality of batches of mesenchymal stem cells in example 5.
Detailed Description
The invention discloses a method for evaluating biological efficacy of mesenchymal stem cells by using CD273, and a person skilled in the art can use the content of the invention to appropriately improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention provides a method for evaluating biological efficacy of mesenchymal stem cells by using CD273, which comprises the following specific evaluation methods:
1. compared with the traditional experimental method comprising collecting stem cell supernatant and detecting cytokines by ELISA method, the method directly adopts stem cell mild digestive enzyme digestion to collect cell marker CD273 antibody for incubation, and obtains the positive rate and average fluorescence intensity MFI of CD273 by flow cytometry. The invention omits complicated steps of collecting culture supernatant, split charging, freezing, re-thawing, ELISA detection and the like, avoids the problem of easy degradation due to unstable cytokine, has simpler and more convenient operation steps, saves time and labor, is more economic and efficient in time, simultaneously avoids the possible human pollution problem, has more stable membrane protein index than cytokine index, is not easy to degrade and is easy to detect. The method can be applied to a main cell bank, a working cell bank and a finished product stage, the sampled sample is directly detected, and the detection result can represent the real condition of the sample. The invention mainly uses a simpler method of analyzing the expression of CD273 by the flow cytometry to carry out correlation analysis with the result of the traditional detection method, and finds out high positive correlation, so that the traditional ELISA method is replaced by the flow mode.
2. The invention firstly illustrates that the expression level of CD273 on the surface of mesenchymal stem cells is obviously and positively related to the biological efficacy level (secretion level of anti-inflammatory factors PGE2 and IDO, inhibition rate of PBMC secreting TNF-alpha and IFN-gamma, inhibition rate of lymphocyte proliferation and Treg promotion rate) within a certain range, and adopts a method of detecting CD273 positive rate and MFI by flow cytometry for the first time to evaluate the biological efficacy of umbilical mesenchymal stem cells.
CD273 is highly and stably expressed under a variety of culture conditions including defined medium, serum (FBS) containing medium and platelet lysate containing medium.
The invention provides a method for evaluating biological effectiveness of mesenchymal stem cells by using CD273, which comprises the steps of marking membrane protein CD273 on the surface of the mesenchymal stem cells by using an antibody, detecting the positive rate of CD273 and average fluorescence intensity MFI of cells by using a flow cytometer, and evaluating the biological effectiveness of the mesenchymal stem cells (CD 273 positive rate is more than 90%, and when CD273-MFI is more than 2000, the inhibition rate of TNF alpha is more than 60% and the inhibition rate of IFN-gamma is more than 80%) by detecting the expression level of CD273 on the surface of the mesenchymal stem cells, wherein the biological effectiveness refers to the secretion level of anti-inflammatory factors PGE2 and IDO, the inhibition rate of PBMC secretion TNF-alpha and IFN-gamma, and the lymphocyte proliferation inhibition rate and Treg promotion rate. The mesenchymal stem cells are umbilical cord, bone marrow, placenta or adipose-derived mesenchymal stem cells.
1. A method for evaluating biological efficacy of mesenchymal stem cells by using CD 273.
2. The mesenchymal stem cell sources include, but are not limited to, umbilical cord, bone marrow, placenta or fat, dental pulp, synovium, etc.
The invention firstly demonstrates that the expression level of CD273 on the surface of mesenchymal stem cells is obviously and positively correlated with the biological efficacy level (secretion level of anti-inflammatory factors PGE2 and IDO, inhibition rate of PBMC secreting TNF-alpha and IFN-gamma, inhibition rate of lymphocyte proliferation and Treg promotion rate) within a certain range, and the biological efficacy of umbilical mesenchymal stem cells is evaluated by adopting a method of detecting CD273 positive rate and MFI by flow cytometry for the first time.
CD273 is highly and stably expressed under a variety of culture conditions including defined medium, serum (FBS) containing medium and platelet lysate containing medium.
Component-limited cultures (basal medium), purchased Yu Youkang organisms, accession number NC0103 (basal medium) +nc0103.s (additive);
FBS-containing medium represents 1640+10% FBS medium, 1640 purchased from imperatorin, cat No. 21870-076; FBS is purchased from Yingweiji, product number 10091-148;
PRP-containing medium (basal medium+platelet lysate), purchased from daceae as company; wherein, 50ml of platelet lysate is added to 500ml of basal medium.
The mesenchymal stem cells in the invention are from umbilical cord.
The raw materials and reagents used in the method for evaluating the biological efficacy of mesenchymal stem cells by using CD273 provided by the invention are all available in the market.
The invention is further illustrated by the following examples:
Example 1
The positive rate of the surface CD273 of the mesenchymal stem cells from different people and the average fluorescence intensity level thereof are detected, and the specific implementation steps are as follows:
flow cytometric detection of expression level of CD273 on the surface of multi-sample umbilical mesenchymal stem cells:
Digestion: the cells of the same batch were discarded, the supernatant was washed with 10mL of physiological saline, the supernatant was removed, the cells were digested at room temperature for 2-3min by adding 4mL/T175 bottle TRYPLE SELECT (gibico), the digestion was stopped by adding 8mL of complete medium for umbilical cord mesenchymal stem cells, the cell suspension was transferred to a 50mL centrifuge tube, 400g,8min centrifugation. The supernatant was discarded and 1-2mL of umbilical cord mesenchymal stem cells were resuspended in complete medium. The umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cell culture conditions comprise specific experimental steps of collecting umbilical cords of healthy puerpera under different culture conditions (adopting composition limiting culture medium, FBS-containing culture medium and PRP-containing culture medium) are collected, the collection process is examined and approved by a hospital ethical committee, and an informed consent form and a health questionnaire are signed with puerpera, so that the puerpera is free of infectious diseases and familial genetic diseases, the Wharton's jelly is separated, primary separation of MSCs is carried out by a tissue block adherence method, and subculture is carried out according to the density of 8000-10000/cm 2, the MSCs are subcultured to 2 generations as seed bank cells for freezing, and are cultured to 4-5 generations for related study, and each cell is transferred into a flow tube according to 5 multiplied by 10 5 cells/tube, and each cell is prepared into a sample tube and a isotype control two tubes. After centrifugation at 1500rpm/5min, cell pellet was left, after washing with physiological saline for 2 times, 100. Mu.l of heavy suspension was added with PE-IgG isotype control antibody and PE-CD273 antibody (Biolgend), and vortexing was performed, incubated at 4℃in the dark for 30min, cells were washed with physiological saline, washed with 1500rpm/5min for 2 times, nonspecific binding was removed, 500. Mu.l of physiological saline was resuspended cells, positive rate of CD273 was detected by 1h internal flow cytometer and MFI and correlation of both were calculated, and the results were shown in FIG. 1, table 1. As can be seen from fig. 1: there was a significant individual variability in the positive rate and MFI of CD273 for a number of different human-derived umbilical cord mesenchymal stem cells, with an average positive rate of 83.4% ± 10.2% for CD273 and an MFI of 2167.8 ± 587.5 for CD 273. Spearman correlation analysis (1B) was performed on the positive rate of cell surface CD273 and MFI, suggesting a significant positive correlation (r=0.8269, p < 0.0001) between the two.
TABLE 1
Example 2
Multiple batches of mesenchymal stem cells were evaluated for factor secretion function and correlation analysis was performed in combination with their CD273 expression levels. The specific implementation method comprises the following steps:
(1) Flow cytometric detection of expression levels of CD273 on the surface of multi-sample mesenchymal stem cells:
flow cytometric detection of expression level of CD273 on the surface of multi-sample umbilical mesenchymal stem cells:
Umbilical mesenchymal stem cells under different culture conditions were collected by digestion (same as in example 1) and transferred into flow tubes at 5X 10 5 cells/tube, each cell being prepared into two tubes of sample tube and isotype control. After centrifugation at 1500rpm/5min, cell pellet was left, after washing with physiological saline 2 times, 100. Mu.l of resuspended PE-IgG isotype control antibody and PE-CD273 antibody (Biolgend) were added, and vortexed and mixed well, incubated at 4deg.C in the dark for 30min, washed with physiological saline for 2 times at 1500rpm/5min, nonspecific binding was removed, 500. Mu.l of physiological saline resuspended cells, and the positive rate of CD273 was detected by 1h internal flow cytometer.
(2) Factor secretion detection of multiple batches of umbilical cord mesenchymal stem cells:
Co-culture experiments: umbilical cord mesenchymal stem cells were counted by digestion, resuspended in 1640 complete medium, plated in 6 well plates at 2X 10 5 cells/2 mL per well, and the plates were evenly spread under microscopic observation and placed in a 5% CO 2 incubator. Peripheral Blood Mononuclear Cells (PBMCs) were resuspended in 1640 complete medium (10. Mu.g/mL PHA added). Wherein the step of activating the PBMCs comprises: 1640+10% FBS+10 μg/mL-PHA was induced for 72h. After 4-6 hours, the umbilical cord mesenchymal stem cells were attached, the activated PBMCs suspension was added to MSCs at a concentration of 2X 10 6 cells/2 mL per well, mixed again, and the control group was PHA-activated PBMC (PBMC group), and the experimental group was co-cultured with PHA-activated PBMC (MSCSs co-culture group). After 72h of incubation, the supernatant was collected and centrifuged at 2500rpm/10min, the cell culture supernatant was aspirated and split into 1 mL/tube frozen at-80℃in preparation for detection of PGE2, IDO, IFN-gamma and TNF-alpha content in the supernatant.
The expression levels in the supernatants were tested with human IFN-gamma and TNF-alpha ELISA kits (abcam) and the inhibition (%) was calculated according to the following formula:
IFN-gamma inhibition (%) = [ 1-IFN-gamma (experimental group)/IFN-gamma (control group) ]. 100%
TNF- α inhibition (%) = [1-TNF- α (experimental group)/TNF- α (control group) ], 100%
(3) The correlation analysis of the CD273 expression level of umbilical cord mesenchymal stem cells and the PGE2 and IDO secretion levels and the inhibition rate of activated PBMC secretion IFN-gamma and TNF-alpha were carried out, and the analysis results are shown in FIG. 2, table 2 and FIG. 3, and Table 3. As can be seen from FIG. 2, there was an individual difference in the secretion amounts of PGE2 and IDO from umbilical cord mesenchymal stem cells of a plurality of different human sources, the average secretion amount of PGE2 was 43.69.+ -. 118.76 (ng/mL), and the average secretion amount of IDO was 18.5.+ -. 9.1 (ng/mL). Spearman correlation analysis was performed on the positive rate of cell surface CD273 with PGE2, IDO secretion, respectively (fig. 2). The results suggest that both are significantly positively correlated. As can be seen from fig. 3: multiple batches of umbilical cord mesenchymal stem cells were co-cultured with activated PBMC for 72 hours, respectively, and the co-culture supernatants were collected to examine the amounts of TNF- α and IFN- γ therein and calculate their inhibition rates for activated PBMC secreting TNF- α (A shown in FIG. 3) and IFN- γ (B shown in FIG. 3), with the TNF- α inhibition rate being (75.94.+ -. 9.68)%, and the IFN- γ inhibition rate being (83.27.+ -. 6.38)%. As a result of spearman correlation analysis of the positive rates of CD273 of umbilical cord mesenchymal stem cells of a plurality of batches and the inhibition rates of the CD273 on activated PBMC secreting IFN-gamma and TNF-alpha, the positive rates of CD273 are obviously and positively correlated with the inhibition rates of TNF-alpha and IFN-gamma respectively (figure 3).
TABLE 2
TABLE 3 Table 3
Example 3
The immunoregulatory function of umbilical cord mesenchymal stem cells of a plurality of batches is evaluated, and correlation analysis is performed in combination with the expression level of CD 273. The specific implementation method comprises the following steps:
(1) Flow cytometric testing of expression levels of CD273 on the surface of umbilical cord mesenchymal stem cells of multiple batches:
flow cytometric testing of expression levels of CD273 on the surface of multi-sample umbilical cord-derived umbilical cord mesenchymal stem cells:
Umbilical cord mesenchymal stem cells under different culture conditions were collected by digestion and transferred into flow tubes at 5X 10 5 cells/tube, each cell being prepared into two tubes of sample tube and isotype control. After centrifugation at 1500rpm/5min, cell pellet was left, after washing with physiological saline 2 times, 100. Mu.l of resuspended PE-IgG isotype control antibody and PE-CD273 antibody (Biolgend) were added, and vortexed and mixed well, incubated at 4deg.C in the dark for 30min, washed with physiological saline for 2 times at 1500rpm/5min, nonspecific binding was removed, 500. Mu.l of physiological saline resuspended cells, and the positive rate of CD273 was detected by 1h internal flow cytometer.
(2) Performing immunoregulatory function detection on a plurality of batches of cells:
Lymphocyte proliferation inhibition assay: umbilical cord mesenchymal stem cells were counted by digestion, resuspended in 1640 complete medium, plated in 6 well plates at 2X 10 5 cells/2 mL per well, and the plates were evenly spread under microscopic observation and placed in a 5% CO 2 incubator. Peripheral Blood Mononuclear Cells (PBMCs) were resuspended in 1640 complete medium (10. Mu.g/mL PHA added). Wherein the step of activating the PBMCs comprises: 1640+10% FBS+10 μg/mL-PHA was induced for 72h. After 4-6 hours, the umbilical cord mesenchymal stem cells are attached, the PBMCs suspension is added into MSCs according to the ratio of 2X 10 6 cells/2 mL per hole, and the mixture is uniformly mixed again, the control group is PHA activated PBMC, and the experimental group is co-culture of the umbilical cord mesenchymal stem cells and the PHA activated PBMC. Supernatants were collected after 72h incubation and centrifuged at 2500rpm/10min for flow detection and cell counts were used for CD3 surface antibody (Biolegend, 300441) labeling and Ki67 intranuclear antibody (Biolegend, 350514) labeling of PBMC (control), co-culture (experimental) groups, respectively. And the inhibition (%) was calculated according to the following formula:
lymphocyte proliferation inhibition ratio (%) = (Ki 67 expression level & control group-Ki 67 expression level & experimental group)/Ki 67 expression level & control group×100%. From fig. 4, table 4 shows that: after the umbilical cord mesenchymal stem cells of a plurality of batches are respectively co-cultured with the activated PBMC for 72 hours, the lymphocyte proliferation inhibition rate is (85.7+/-6.75)%, and after the analysis of spearman correlation between the positive rate of the umbilical cord mesenchymal stem cells CD273 of a plurality of batches and the lymphocyte proliferation inhibition rate thereof, the positive rate of the CD273 is found to be obviously and positively correlated with the lymphocyte proliferation inhibition rate respectively.
TABLE 4 Table 4
Treg promotion experiment: umbilical cord mesenchymal stem cells were counted by digestion, resuspended in 1640 complete medium, plated in 6 well plates at 2X 10 5 cells/2 mL per well, and the plates were evenly spread under microscopic observation and placed in a 5% CO 2 incubator. PBMC were activated with 1640 complete medium+10% FBS+2 μg/mLCD3 activated antibody (Bori doctor biotechnology Co., ltd., T210) +2 μg/mL CD28 activated antibody (Beijing Baiposi Biotechnology Co., ltd., CD8-M120 b) +1500IU/mL IL 2. After 4-6 hours, umbilical mesenchymal stem cells were attached, the complete medium of 1640+10% FBS+2 μg/mLCD3 activated antibody+2 μg/mL CD28 activated antibody was used as per 2×10 6 cells/2 mL per well
+1500IU/mL IL2, 72h activated PBMCs suspension added to MSCs, again mixed, control group for activated PBMC, experimental group for umbilical cord mesenchymal stem cells and activated PBMC co-culture. Supernatants were collected after 72h incubation, and supernatants were collected after 72h incubation and centrifuged at 2500rpm/10min for flow detection and cell counts were used for CD4 surface antibody (Biolegend, 344604) labeling, CD25 surface antibody (Biolegend, 302610) labeling and FOXP3 intranuclear antibody (Biolegend, 364704) labeling, respectively, for PBMC (control), co-culture (experimental). And calculating Treg promotion rate (%) according to the following formula: treg promotion (%) = [ Treg (experimental group) -Treg (control group)/Treg (experimental group) ]x100%.
From fig. 5, table 5 shows that: after the umbilical cord mesenchymal stem cells of a plurality of batches are respectively co-cultured with the activated PBMC for 72 hours, the Treg promotion rate is (122+/-24)%, and after the positive rate of CD273 and the lymphocyte proliferation inhibition rate of the umbilical cord mesenchymal stem cells of a plurality of batches are subjected to spearman correlation analysis, the positive rate of CD273 is obviously positively correlated with the lymphocyte proliferation inhibition rate respectively.
TABLE 5
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
- Use of cd273 in the preparation of a product for evaluating the biological efficacy of mesenchymal stem cells.
- 2. The use of claim 1, wherein said evaluating the biological efficacy of mesenchymal stem cells comprises detecting any of:(1) Anti-inflammatory factor content; and/or(2) Inhibition of cytokines; and/or(3) Lymphocyte proliferation inhibition rate and/or Treg promotion rate.
- 3. The use according to claim 1 or 2, wherein the anti-inflammatory factor comprises PGE2 and/or IDO;the cytokines include TNF-alpha and/or IFN-gamma.
- 4. The use according to any one of claims 1 to 3, wherein the expression level of CD273 is significantly positively correlated with the level of biological efficacy of the mesenchymal stem cells.
- 5. The use according to any one of claims 1 to 4, wherein the criteria for the evaluation are: when the CD273 positive rate is more than 90%, the TNF-alpha inhibition rate of the mesenchymal stem cells is more than 60% and/or the IFN-gamma inhibition rate is more than 80%.
- 6. The use according to any one of claims 1 to 5, wherein the source of mesenchymal stem cells comprises one or more of umbilical cord, bone marrow, placental fat, dental pulp or synovium.
- 7. A method for evaluating biological efficacy of mesenchymal stem cells by using CD273, which is characterized in that the mesenchymal stem cells after culture are incubated with CD273 antibody, and the biological efficacy of the mesenchymal stem cells is obtained by detecting the CD273 positive rate and MFI by using flow cytometry.
- 8. The method of claim 7, wherein said evaluating mesenchymal stem cell biological efficacy comprises detecting any of:(1) Anti-inflammatory factor content; and/or(2) Inhibition of cytokines; and/or(3) Lymphocyte proliferation inhibition rate and/or Treg promotion rate;the anti-inflammatory factors include PGE2 and/or IDO;the cytokines include TNF-alpha and/or IFN-gamma.
- 9. The method according to any one of claims 7 to 8, wherein the criteria for the evaluation are: when the CD273 positive rate is more than 90%, the TNF-alpha inhibition rate of the mesenchymal stem cells is more than 60% and/or the IFN-gamma inhibition rate is more than 80%.
- 10. The method of any one of claims 7 to 9, wherein the culture medium employed in the culturing comprises one or more of a defined medium, an FBS-containing medium, or a platelet lysate-containing medium;the source of the mesenchymal stem cells comprises one or more of umbilical cord, bone marrow, placental fat, dental pulp or synovium.
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