Method for screening umbilical cord mesenchymal stem cells for effectively treating myocardial infarction by using N-cdherin+ / CD168-umbilical cord mesenchymal stem cell subpopulation

Abstract

The invention provides a method for screening umbilical cord mesenchymal stem cells capable of effectively treating myocardial infarction by using an N-cadhein < + > / CD168 <-> umbilical cord mesenchymal stem cell subset. The method comprises the following steps: obtaining umbilical cord mesenchymal stem cells; the umbilical cord mesenchymal stem cells are incubated with an N-cadhein-PE antibody and a CD168-FITC antibody, and the incubated umbilical cord mesenchymal stem cells are obtained; and detecting the ratio of the N-cadhein + / CD168-umbilical cord mesenchymal stem cell subpopulation in the incubated umbilical cord mesenchymal stem cells, and predicting the effectiveness of the umbilical cord mesenchymal stem cells in the treatment of myocardial infarction according to the ratio: when the ratio is greater than 25%, predicting that the umbilical cord mesenchymal stem cells have the effect of effectively treating myocardial infarction. By adopting the method provided by the invention, the umbilical cord mesenchymal stem cells meeting the standard of effectively treating myocardial infarction can be obtained, and the method has important medical value and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of cell therapy, in particular to a method of using N-cadherin + / CD168 - Method for screening umbilical cord mesenchymal stem cells subpopulations for effective treatment of myocardial infarction. Background Art

[0002] Myocardial infarction (MI), also known as myocardial infarction, is caused by the occlusion of coronary arteries, which leads to the death of a large number of myocardial cells, resulting in impaired heart function and progressive heart failure. If not treated in time, myocardial infarction may lead to cardiac arrest and death. Therefore, it is crucial to find effective treatments to prevent or treat myocardial infarction.

[0003] Mesenchymal stem cells (MSCs) are multipotent stem cells derived from mature tissues, with high self-renewal ability and multidirectional differentiation potential, and are new seed cells for cell transplantation and tissue engineering. Animal experiments and clinical trials have shown that mesenchymal stem cells (MSCs) have low immunogenicity and paracrine effects, so transplantation of mesenchymal stem cells has become a promising therapeutic strategy for the treatment of myocardial infarction.

[0004] The mesenchymal stem cells obtained so far meet the minimum standards of the International Association for Cellular Therapy: (1) The cell morphology is similar to that of fibroblasts; (2) The cells show surface molecular characteristics of CD73 positive, CD90 positive, and CD105 positive, and at the same time CD14 negative or CD1b negative, CD19 negative or CD79α negative, CD34 negative, CD45 negative, and HLA-DR negative; (3) They have the potential for trilineage differentiation under in vitro culture conditions. This standard is widely accepted and adopted in basic research and industrial applications. However, mesenchymal stem cells from different sources have different characteristics. This standard only defines the basic morphological and functional characteristics of mesenchymal stem cells. Therefore, the mesenchymal stem cells under this standard are a group of related but not completely identical heterogeneous cell populations, and cannot screen out mesenchymal stem cells that meet the indicators for effective treatment of myocardial infarction. Summary of the invention

[0005] The present invention provides a method for utilizing N-cadherin + / CD168 - Method for screening umbilical cord mesenchymal stem cells subpopulations for effective treatment of myocardial infarction by detecting N-cadherin + / CD168 -The ratio of umbilical cord mesenchymal stem cell subpopulations in umbilical cord mesenchymal stem cells, screening out N-cadherin + / CD168 - Umbilical cord mesenchymal stem cells with a subpopulation ratio of umbilical cord mesenchymal stem cells greater than 25% are selected, thereby establishing an effectiveness index for screening umbilical cord mesenchymal stem cells that are effective in treating myocardial infarction.

[0006] The present invention also provides umbilical cord mesenchymal stem cells screened and obtained by the above method, wherein the umbilical cord mesenchymal stem cells can improve the ejection fraction and short-axis shortening rate of the heart of an individual after myocardial infarction, reduce the left ventricular end-systolic diameter and left ventricular end-diastolic diameter of the heart of an individual after myocardial infarction, reduce the area of ​​cardiac damage, and significantly improve the cardiac function of an individual after myocardial infarction.

[0007] The present invention also provides a stem cell therapeutic drug for myocardial infarction, which contains the above-mentioned umbilical cord mesenchymal stem cells. The stem cell therapeutic drug for myocardial infarction can effectively treat myocardial infarction.

[0008] The first aspect of the present invention provides a method for utilizing N-cadherin + / CD168 - The method for screening umbilical cord mesenchymal stem cells subpopulations for effectively treating myocardial infarction comprises:

[0009] Obtaining umbilical cord mesenchymal stem cells;

[0010] Incubating the umbilical cord mesenchymal stem cells with N-cadherin-PE antibody and CD168-FITC antibody to obtain incubated umbilical cord mesenchymal stem cells;

[0011] Detection of N-cadherin + / CD168 - The ratio of the umbilical cord mesenchymal stem cell subpopulation in the incubated umbilical cord mesenchymal stem cells, and the effectiveness of the umbilical cord mesenchymal stem cells in treating myocardial infarction is predicted according to the ratio:

[0012] When the ratio is greater than 25%, it is predicted that the umbilical cord mesenchymal stem cells have an effective effect in treating myocardial infarction.

[0013] In the above method, the number of the umbilical cord mesenchymal stem cells is (2-5)×10 5 indivual.

[0014] In the method described above, the volume of the N-cadherin-PE antibody is 5 μl to 10 μl, and the volume of the CD168-FITC antibody is 1 μl to 2 μl.

[0015] In the above method, the specific process of incubation is:

[0016] The umbilical cord mesenchymal stem cells are subjected to a first centrifugation, and then the umbilical cord mesenchymal stem cells after the first centrifugation are subjected to a first washing with a first PBS buffer, and the supernatant is removed after the first washing to obtain an umbilical cord mesenchymal stem cell precipitate; a cell staining buffer, an N-cadherin-PE antibody, and a CD168-FITC antibody are added to the umbilical cord mesenchymal stem cell precipitate for incubation, and after incubation, a second centrifugation is performed and the supernatant is removed, and a second PBS buffer is added to obtain the incubated umbilical cord mesenchymal stem cells.

[0017] According to the method described above, the first centrifugation speed is 1000r / min to 1500r / min and the time is 3min to 5min;

[0018] And / or, the first cleaning is performed 2 to 5 times;

[0019] And / or, the incubation temperature is 0°C to 4°C and the time is 20min to 30min;

[0020] And / or, the second centrifugation has a rotation speed of 1000 r / min to 1500 r / min and a time of 3 min to 5 min.

[0021] In the method described above, the volume of the first PBS buffer is 2 mL to 5 mL;

[0022] and / or, the volume of the cell staining buffer is 100 μl to 200 μl;

[0023] And / or, the volume of the second PBS buffer is 500 μl to 1000 μl.

[0024] The method as described above further comprises pre-treating the umbilical cord mesenchymal stem cells before incubating, and the specific process of the pre-treatment is:

[0025] The umbilical cord mesenchymal stem cells are inoculated into a culture medium and cultured for 4 to 5 days, and then the culture medium is removed; the umbilical cord mesenchymal stem cells are then washed and digested for a second time and then added to the culture medium; the supernatant is removed after a third centrifugation to obtain a cell precipitate; the cell precipitate is resuspended to obtain the pretreated umbilical cord mesenchymal stem cells.

[0026] The method as described above, wherein the culture medium is a culture medium containing 5 vol% fetal bovine serum, 1 vol% double antibody and 1 vol% mesenchymal stem cell additive;

[0027] And / or, the third centrifugation has a rotation speed of 1000 r / min to 1500 r / min and a time of 3 min to 5 min.

[0028] The second aspect of the present invention provides umbilical cord mesenchymal stem cells obtained by screening according to the method.

[0029] The third aspect of the present invention provides a stem cell treatment drug for myocardial infarction, comprising the umbilical cord mesenchymal stem cells.

[0030] The present invention provides a method for utilizing N-cadherin + / CD168 - Method for screening umbilical cord mesenchymal stem cells subpopulations for effective treatment of myocardial infarction by detecting N-cadherin + / CD168 - The ratio of umbilical cord mesenchymal stem cell subpopulations in umbilical cord mesenchymal stem cells after incubation is used to predict the effectiveness of umbilical cord mesenchymal stem cells in treating myocardial infarction: when the ratio is greater than 25%, it is predicted that umbilical cord mesenchymal stem cells have an effective effect in treating myocardial infarction. The umbilical cord mesenchymal stem cells screened by this method can improve the ejection fraction and short-axis shortening rate of the heart of an individual after myocardial infarction, reduce the left ventricular end-systolic diameter and left ventricular end-diastolic diameter of the heart of an individual after myocardial infarction, reduce the area of ​​cardiac damage, and significantly improve the cardiac function of an individual after myocardial infarction. Therefore, the method provided by the present invention can screen and obtain umbilical cord mesenchymal stem cells that meet the standards for effective treatment of myocardial infarction, which has important medical and economic value. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 is the flow cytometry histogram of the surface molecular characteristics of MSC-GY;

[0033] in, Figure 1 A is the flow cytometry histogram of CD44;

[0034] B is the flow cytometry histogram of CD90;

[0035] C is the flow cytometry histogram of CD105;

[0036] D is the flow cytometry histogram of CD73;

[0037] E is the flow cytometry histogram of CD34;

[0038] F is the flow cytometry histogram of HLA-DR.

[0039] Figure 2 The tSNE diagram shows the four cell subpopulations and statistical results of MSC-GY.

[0040] Figure 3 Bubble chart showing gene enrichment analysis of differentially expressed genes in the four cell subsets of MSC-GY.

[0041] Figure 4 tSNE plot in MSC-GY identified by Scissor;

[0042] in, Figure 4 A is the tSNE diagram of Scissor+ and Scissor- cells in MSC-GY; B is the tSNE diagram of Scissor + Statistical results of cells in four cell subsets.

[0043] Figure 5 N-cadherin in MSC-GY + and CD168 - tSNE plot of

[0044] in, Figure 5 A is the tSNE diagram of N-cadherin;

[0045] B is the tSNE diagram of CD168.

[0046] Figure 6 is the flow cytometry histogram of the surface molecular characteristics of MSC-CX;

[0047] in, Figure 6 A is the flow cytometry histogram of CD44;

[0048] B is the flow cytometry histogram of CD90;

[0049] C is the flow cytometry histogram of CD105;

[0050] D is the flow cytometry histogram of CD73;

[0051] E is the flow cytometry histogram of CD34;

[0052] F is the flow cytometry histogram of HLA-DR.

[0053] Figure 7 Flow cytometry plot of surface molecular characteristics of MSC-CJ;

[0054] in, Figure 7 A is the flow cytometry plot of CD44;

[0055] B is the flow cytometry histogram of CD90;

[0056] C is the flow cytometry histogram of CD105;

[0057] D is the flow cytometry histogram of CD73;

[0058] E is the flow cytometry histogram of CD34;

[0059] F is the flow cytometry histogram of HLA-DR.

[0060] Figure 8 N-cadherin + / CD168 - Flow cytometric analysis scatter plots of cell subpopulations;

[0061] in, Figure 8 A is the presentation of N-cadherin in MSC-CX + / CD168 - Flow cytometric analysis scatter plots of cell subpopulations;

[0062] B: Presentation of N-cadherin in MSC-CJ + / CD168 - Flow cytometric analysis scatter plots of cell subpopulations;

[0063] C shows N-cadherin in MSC-GY + / CD168 - Flow cytometric analysis scatter plots of cell subpopulations;

[0064] D is the expression of N-cadherin in MSC-CX, MSC-CJ, and MSC-GY + / CD168 - Flow cytometric analysis of cell subpopulation ratios.

[0065] Fig. 9 These are the results of small animal ultrasound examination of the hearts of mice with myocardial infarction in each group 4 weeks later;

[0066] in, Fig. 9 A is the ejection fraction test result of the heart of each group of myocardial infarction mice 4 weeks later;

[0067] B is the test result of the left ventricular end-systolic diameter of the hearts of mice with myocardial infarction in each group 4 weeks later;

[0068] C is the test result of the short axis shortening rate of the hearts of mice with myocardial infarction in each group 4 weeks later;

[0069] D is the test result of the left ventricular end-diastolic diameter of the hearts of mice with myocardial infarction in each group 4 weeks later.

[0070] Fig.10The results of Masson's trichrome staining of heart tissues of mice with myocardial infarction in each group 4 weeks later;

[0071] in, Fig.10 A is the Masson's trichrome staining result of serial sections of heart tissue of mice with myocardial infarction in each group 4 weeks later (one section of heart tissue was taken every 500 μm for Masson's trichrome staining, and a total of 5 sections were taken for each heart tissue);

[0072] B is the statistical result of Masson's trichrome staining of the damaged area of ​​heart tissue in mice with myocardial infarction 4 weeks later. DETAILED DESCRIPTION

[0073] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. If the specific technology or conditions are not indicated in the embodiments, the technology or conditions described in the literature in this area or the product instructions are carried out. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0074] It should be noted that, in the present invention, there are descriptions involving “first”, “second”, etc., and the descriptions of “first”, “second”, etc. are only used for descriptive purposes and therefore cannot be understood as limitations on the present invention.

[0075] As used herein, the term "treat" refers to ameliorating a disease or condition (i.e., slowing down or preventing or alleviating the development of a disease or at least one of its clinical symptoms) in some embodiments. In other embodiments, "treat" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treat" refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In other embodiments, "treat" refers to preventing or delaying the onset, occurrence, or worsening of a disease or condition.

[0076] The term "and / or" used in the present invention is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. Among them, A and B can be singular or plural; the single symbol " / " means "or".

[0077] The first aspect of the present invention provides a method for utilizing N-cadherin + / CD168- The method for screening umbilical cord mesenchymal stem cells subpopulations for effectively treating myocardial infarction comprises:

[0078] Obtaining umbilical cord mesenchymal stem cells;

[0079] The umbilical cord mesenchymal stem cells are incubated with N-cadherin-PE antibody and CD168-FITC antibody to obtain the incubated umbilical cord mesenchymal stem cells;

[0080] Detection of N-cadherin + / CD168 - The ratio of umbilical cord mesenchymal stem cell subpopulations in the umbilical cord mesenchymal stem cells after incubation, according to which the effectiveness of umbilical cord mesenchymal stem cells in treating myocardial infarction is predicted:

[0081] When the ratio is greater than 25%, it is predicted that umbilical cord mesenchymal stem cells have an effective effect in treating myocardial infarction.

[0082] The inventors of the present invention conducted in-depth research on umbilical cord mesenchymal stem cells and found that umbilical cord mesenchymal stem cells contain N-cadherin + / CD168 - Cell subsets, the N-cadherin + / CD168 - The cell subpopulation is the key cell in umbilical cord mesenchymal stem cells that can effectively improve the cardiac function of individuals after myocardial infarction. Of course, it can be understood by those skilled in the art that they can classify umbilical cord mesenchymal stem cells into subpopulations according to any known surface molecules.

[0083] Furthermore, the inventors have found through research that when N-cadherin + / CD168 - When the ratio of the umbilical cord mesenchymal stem cell subpopulation in the umbilical cord mesenchymal stem cells after incubation is greater than 25%, the umbilical cord mesenchymal stem cells can significantly improve the cardiac function of individuals after myocardial infarction, and it is predicted that the umbilical cord mesenchymal stem cells have an effective effect in treating myocardial infarction. + / CD168 - The umbilical cord mesenchymal stem cells obtained by using the umbilical cord mesenchymal stem cell subpopulation ratio greater than 25% as a classification index can effectively improve the cardiac function of individuals after myocardial infarction.

[0084] The present invention uses flow cytometry analysis technology to detect N-cadherin + / CD168 - The ratio of umbilical cord mesenchymal stem cell subpopulations in umbilical cord mesenchymal stem cells after incubation.

[0085] The expression "myocardial infarction" used here refers to the massive death of myocardial cells due to coronary artery occlusion, which leads to impaired cardiac function and progressive heart failure. A person skilled in the art can know that an individual has the above-mentioned myocardial infarction through routine tests, and of course can also determine whether the individual's cardiac function is improved after myocardial infarction.

[0086] The N-cadherin-PE antibody and CD168-FITC antibody used in the present invention are molecular markers for incubating umbilical cord mesenchymal stem cells. The present invention uses N-cadherin-PE antibody and CD168-FITC antibody to incubate umbilical cord mesenchymal stem cells. N-cadherin-PE antibody and CD168-FITC antibody can bind to the corresponding antigens on the surface of umbilical cord mesenchymal stem cells, and can effectively identify N-cadherin with specific molecular markers. + / CD168 - Umbilical cord mesenchymal stem cell subsets (N-cadherin + / CD168 - cell subsets), which is beneficial for subsequent detection of N-cadherin + / CD168 - Umbilical cord mesenchymal stem cell subset ratio (N-cadherin + / CD168 - cell subset ratios).

[0087] Further, the ratio may preferably be greater than 29.4%.

[0088] In the present invention, the number of umbilical cord mesenchymal stem cells is (2-5)×10 5 indivual.

[0089] When the number of umbilical cord mesenchymal stem cells is within the above range, after removing adhesion cells and dead cells during the detection process, a sufficient number of single umbilical cord mesenchymal stem cells can be obtained in a relatively short time, which can accurately represent the characteristics of the overall cells.

[0090] In the present invention, the volume of the N-cadherin-PE antibody is 5 μl to 10 μl, and the volume of the CD168-FITC antibody is 1 μl to 2 μl.

[0091] When the volume of the N-cadherin-PE antibody and the volume of the CD168-FITC antibody are within the above range, the N-cadherin-PE antibody and the CD168-FITC antibody can fully bind to the antigens on the surface of umbilical cord mesenchymal stem cells, saving antibody costs and improving the accuracy of subsequent detection.

[0092] In the present invention, the specific process of incubation is:

[0093] The umbilical cord mesenchymal stem cells are subjected to a first centrifugation, and then the umbilical cord mesenchymal stem cells after the first centrifugation are subjected to a first washing with a first PBS buffer, and the supernatant is removed after the first washing to obtain an umbilical cord mesenchymal stem cell precipitate; a cell staining buffer, an N-cadherin-PE antibody, and a CD168-FITC antibody are added to the umbilical cord mesenchymal stem cell precipitate for incubation, and after incubation, a second centrifugation is performed and the supernatant is removed, and a second PBS buffer is added to obtain the incubated umbilical cord mesenchymal stem cells.

[0094] The present invention improves the positive rate of umbilical cord mesenchymal stem cells by fully incubating the umbilical cord mesenchymal stem cells with N-cadherin-PE antibody and CD168-FITC antibody.

[0095] In the present invention, the speed of the first centrifugation is 1000r / min to 1500r / min, and the time is 3min to 5min. The number of first washings is 2 to 5 times. The temperature of incubation is 0°C to 4°C, and the time is 20min to 30min. The speed of the second centrifugation is 1000r / min to 1500r / min, and the time is 3min to 5min. The volume of the first PBS buffer is 2mL to 5mL. The volume of the cell staining buffer is 100μl to 200μl. The volume of the second PBS buffer is 500μl to 1000μl.

[0096] When the digestion time, the rotation speed and time of the first centrifugation, the number of first washing times, the temperature and time of incubation, the rotation speed and time of the second centrifugation, the volume of the first PBS buffer, the volume of the cell staining buffer and the volume of the second PBS buffer are each within the above ranges, the umbilical cord mesenchymal stem cells can be completely covered and non-specific binding can be removed during the incubation process, and the surviving umbilical cord mesenchymal stem cells can be completely obtained without damage during the second centrifugation process.

[0097] In the present invention, the umbilical cord mesenchymal stem cells are pretreated before incubation, and the specific process of the pretreatment is:

[0098] The umbilical cord mesenchymal stem cells are inoculated into a culture medium and cultured for 4 to 5 days, and then the culture medium is removed; the umbilical cord mesenchymal stem cells are then washed and digested for a second time and then added to the culture medium; the supernatant is removed after a third centrifugation to obtain a cell precipitate; the cell precipitate is resuspended to obtain the pretreated umbilical cord mesenchymal stem cells.

[0099] The present invention pre-treats the umbilical cord mesenchymal stem cells so that the umbilical cord mesenchymal stem cells are fully digested into single cells, thereby ensuring that each cell is evenly incubated, thereby enabling effective flow cytometric analysis and identification.

[0100] The digestive enzyme used in the above digestion includes tryple, and the digestion time is 2 minutes to 3 minutes. The umbilical cord mesenchymal stem cells after the second washing are digested, and the umbilical cord mesenchymal stem cells can be gently digested into single cells.

[0101] In the present invention, the culture medium is a culture medium containing 5 vol% fetal bovine serum, 1 vol% double antibody and 1 vol% umbilical cord mesenchymal stem cell additive. The culture medium can maintain the proliferation characteristics, cell activity, cell morphology, cell purity and differentiation induction ability of umbilical cord mesenchymal stem cells.

[0102] In the present invention, the third centrifugation has a rotation speed of 1000 r / min to 1500 r / min and a time of 3 min to 5 min.

[0103] When the parameters of the rotation speed and time of the third centrifugation are within the above range, the dead cells in the umbilical cord mesenchymal stem cells can be effectively removed, and the surviving single umbilical cord mesenchymal stem cells can be precipitated without affecting their cell characteristics.

[0104] The present invention also includes filtering the incubated umbilical cord mesenchymal stem cells, and the filtering process is: using a filter with a pore size of 40 to 50 μm to filter the incubated umbilical cord mesenchymal stem cells. The filtering process can remove adherent cell clumps and improve the accuracy of the test results.

[0105] The second aspect of the present invention provides umbilical cord mesenchymal stem cells screened and obtained by the above method. The umbilical cord mesenchymal stem cells can improve the ejection fraction and short axis shortening rate of the heart of individuals after myocardial infarction, reduce the area of ​​cardiac damage, and significantly improve the cardiac function of individuals after myocardial infarction.

[0106] The third aspect of the present invention provides a stem cell therapy drug for myocardial infarction, comprising the above-mentioned umbilical cord mesenchymal stem cells. The stem cell therapy drug for myocardial infarction can effectively treat myocardial infarction.

[0107] The following is a method for utilizing N-cadherin provided by the present invention in combination with the embodiments. + / CD168 - The method of screening umbilical cord mesenchymal stem cells subpopulations for effectively treating myocardial infarction is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0108] Example 1: Screening of cell subpopulations related to myocardial infarction treatment in umbilical cord mesenchymal stem cells I. Experimental materials

[0109] Umbilical cord mesenchymal stem cells (MSC-GY) provided by the National Stem Cell Translation Resource Bank.

[0110] 2. Experimental methods and results

[0111] (1) Flow cytometry was used to detect CD44, CD90, CD105, CD73, CD34, and HLA-DR in umbilical cord mesenchymal stem cells (MSC-GY). The test results were as follows: Figure 1 shown.

[0112] (2) Single-cell sequencing (scRNA-seq) of the umbilical cord mesenchymal stem cells (MSC-GY) was performed using the 10×Genomics platform. The single-cell sequencing data results were analyzed using the Seurat package version 4.1.0 in R to obtain a matrix. The top 2000 highly expressed genes were selected using the matrix, and the t-SNE (t-Distributed Stochastic Neighbor Embedding) algorithm was used to reduce the dimension of the data and visualize the results. Figure 2 shown.

[0113] (3) Gene enrichment analysis was performed to analyze the functional enrichment pathways of each cell subset in umbilical cord mesenchymal stem cells (MSC-GY). Figure 3 shown.

[0114] (4) The above single-cell sequencing data were predicted and analyzed using Scissor technology. The results are as follows: Figure 4 shown.

[0115] (5) Identification of the molecular surface characteristics of cell subgroup 1 (Cluster 1). The results are as follows Figure 5 shown.

[0116] 3. Experimental Results

[0117] Figure 1 The results showed that the umbilical cord mesenchymal stem cells (MSC-GY) were CD44 positive (CD44 + ), CD90 positive (CD90 + ), CD105 positive (CD105 + ), CD73 positive (CD73 + ), CD34 negative (CD34 - ) and HLA-DR negative (HLA-DR - ) molecular characteristics, which are consistent with the basic characteristics of mesenchymal stem cells.

[0118] Figure 2The results showed that umbilical cord mesenchymal stem cells mainly included 4 cell subgroups (Cluster 1, Cluster 2, Cluster 3, and Cluster 4). Among them, Cluster 1 contained 3672 cells, accounting for 35.10%; Cluster 2 contained 2914 cells, accounting for 27.85%; Cluster 3 contained 2413 cells, accounting for 23.06%; and Cluster 4 contained 1464 cells, accounting for 13.99%.

[0119] Figure 3 The results showed that the characteristic genes of cell subset 1 (Cluster 1) were mainly enriched in biological processes related to angiogenesis, such as vascular development and vascular remodeling.

[0120] Scissor technology can use phenotypes and scRNA-seq to identify key cell subpopulations related to biology or clinical prognosis from single-cell sequencing data. Scissor technology mainly integrates single-cell data related to phenotype by quantifying the gene expression profile data of each single cell and the gene expression similarity between each single cell. Then, the regression model of the correlation matrix is ​​optimized by phenotype to identify relevant cell subpopulations. The single-cell sequencing data of the above-mentioned umbilical cord mesenchymal stem cells (MSC-GY) were predicted and analyzed by Scissor technology. MSC-M02 is a mesenchymal stem cell that has been reported to significantly increase the short-axis shortening rate and ejection fraction of the left ventricle of rats with myocardial infarction and reduce the infarct area, while MSC-M01 is a mesenchymal stem cell that has been reported to have little effect on myocardial infarction damage (https: / / pubmed.ncbi.nlm.nih.gov / 22068423 / ). Through integrated analysis, Scissor technology identified a total of 1,890 Scissor genes in MSC-GY that are similar to MSC-M02 in expression profile. + cell( Figure 4 The statistical results show that ( Figure 4 B), in 1890 Scissor + Among the cells, 1824 umbilical cord mesenchymal stem cells (96.5%) were from cluster 1, which indicated that cluster 1 was associated with improving cardiac function after myocardial infarction.

[0121] The top 2000 highly expressed genes in cluster 1 were compared with https: / / www.uniprot.org / Intersection analysis of proteins expressed on the cell membrane in human cells provided by Figure 5Results A and B show that cluster 1 expressed N-cadherin + / CD168 - molecular surface features, so cell subset 1 is N-cadherin + / CD168 - Umbilical cord mesenchymal stem cell subsets (N-cadherin + / CD168 - cell subsets).

[0122] Example 2: Detection of N-cadherin + / CD168 - The proportion of cell subpopulations in umbilical cord mesenchymal stem cells

[0123] 1. Experimental Materials

[0124] Two other umbilical cord mesenchymal stem cells from different donor sources provided by the National Stem Cell Translation Resource Bank: MSC-CX and MSC-CJ; and the umbilical cord mesenchymal stem cells in the above Example 1: MSC-GY.

[0125] 2. Experimental Methods

[0126] (1) MSC-CX and MSC-CJ were tested for CD44, CD90, CD105, CD73, CD34, and HLA-DR by flow cytometry. The results are as follows Figure 6 and Figure 7 shown.

[0127] (2) Detection of N-cadherin + / CD168 - The method for calculating the ratio of cell subpopulations in umbilical cord mesenchymal stem cells is as follows:

[0128] Pretreatment: After the umbilical cord mesenchymal stem cells (sample) were inoculated into a culture medium (ScienCell#7501) and cultured for 5 days, the culture medium was removed, and the umbilical cord mesenchymal stem cells were washed once with PBS buffer (gibco 20012027), and then the umbilical cord mesenchymal stem cells were digested with 2 ml of Tryple (gibco 12604021) for 3 min and then the culture medium (ScienCell#7501) was added, the umbilical cord mesenchymal stem cells were blown into single cells, centrifuged in a 1000 r / min centrifuge for 3 min, and the supernatant was removed to obtain a cell precipitate; 5 ml of PBS buffer (gibco 20012027) was added to the cell precipitate for re-suspending to obtain the pretreated umbilical cord mesenchymal stem cells; the above-mentioned culture medium (ScienCell#7501) is a culture medium containing 5 vol% fetal bovine serum, 1% vol% double antibody and 1 vol% mesenchymal stem cell additive;

[0129] Take the number as 3×10 5 The pretreated umbilical cord mesenchymal stem cells were centrifuged at 1500 r / min for 3 min, and then washed twice with 3 mL PBS buffer (gibco 20012027), and the supernatant was removed to obtain umbilical cord mesenchymal stem cell pellets; 100 μl cell staining buffer (Biolegend 420201), 6 μl N-cadherin-PE (Sino biological: 11039-R001-PE) antibody and 1.5 μl CD168-FITC (Bioss: bs-4736R-FITC) antibody were added to the umbilical cord mesenchymal stem cell pellets for incubation at 4°C in the dark for 30 min, and then centrifuged at 1500 r / min for 3 min, the supernatant was removed, and 500 μl PBS buffer (gibco20012027) was added to obtain incubated umbilical cord mesenchymal stem cells;

[0130] Umbilical cord mesenchymal stem cells were filtered through a filter with a pore size of 40 μm after incubation, and then analyzed by flow cytometry in a BD FACSVerse instrument. N-cadherin was detected by flow cytometry analysis technology + / CD168 - The ratio of cell subpopulations in umbilical cord mesenchymal stem cells after incubation (N-cadherin + / CD168 - Finally, FlowJo was used to calculate the N-cadherin+ / CD168- cell subset ratio.

[0131] MSC-CX, MSC-CJ and MSC-GY were used as samples, and N-cadherin was obtained according to the above method. + / CD168 - The ratio of cell subpopulations in MSC-CX, MSC-CJ and MSC-GY (N-cadherin + / CD168 - The results are as follows Figure 8 As shown in A-C, the N-cadherin + / CD168 - The cell subset ratios, such as Figure 8 As shown in D.

[0132] 3. Experimental Results

[0133] Figure 6 and Figure 7 The results showed that both MSC-CX and MSC-CJ presented CD44 positive (CD44 + ), CD90 positive (CD90 + ), CD105 positive (CD105 + ), CD73 positive (CD73 + ), CD34 negative (CD34 - ) and HLA-DR negative (HLA-DR - ) molecular characteristics, which are consistent with the basic characteristics of mesenchymal stem cells.

[0134] Depend on Figure 8 AD results showed that N-cadherin + / CD168 - The proportion of cell subpopulations in MSC-CX was 17.3%, and N-cadherin + / CD168 - The proportion of cell subpopulations in MSC-CJ was 29.4%, and N-cadherin + / CD168 - The proportion of cell subpopulations in MSC-GY was 34.9%. + / CD168 - The proportions of cell subpopulations in umbilical cord mesenchymal stem cells from different donors are different, and N-cadherin was found + / CD168 -The ratio of cell subpopulations in MSC-CX is lower than that in MSC-GY and MSC-CJ, which predicts that the effects of MSC-GY and MSC-CJ in the treatment of myocardial infarction are significantly higher than those of MSC-CX. MSC-GY and MSC-CJ are effective in treating myocardial infarction.

[0135] Example 3: Validation of different N-cadherins + / CD168 - Effectiveness of umbilical cord mesenchymal stem cells in the treatment of myocardial infarction based on cell subset ratio

[0136] 1. Experimental Methods

[0137] (1) Construction of myocardial infarction mouse model: Healthy C57BL / 6 male mice weighing 30 g were anesthetized using an isopentane gas anesthesia apparatus. The left side of the mouse chest was opened, and the heart was exposed by entering the chest through the 3 / 4 intercostal space of the mouse using an elbow needle holder. The left anterior descending branch of the coronary artery was ligated 3 mm below the left atrial appendage of the mouse using a 7-0 suture needle. The myocardium in the vascular innervation area of ​​the mouse turned white, indicating successful ligation. The chest was closed layer by layer to obtain a mouse with myocardial infarction (MI).

[0138] (2) Experimental groups: sham group (Sham), control group (Ctrl), MSC-CX group (MSC-CX), MSC-GY group (MSC-GY), and MSC-CJ group (MSC-CJ);

[0139] Sham operation group (Sham): healthy C57BL / 6 male mice weighing 30 g were obtained by piercing the heart tissue of the mice with a 7-0 suture needle without ligation. Four weeks later, the hearts of the sham operation mice were subjected to small animal ultrasound examination and Masson's trichrome staining experiments.

[0140] Control group (Ctrl): 25 μl of normal saline was injected into the mice with myocardial infarction, and the heart tissues of the mice with myocardial infarction were subjected to small animal ultrasound detection and Masson's trichrome staining experiments 4 weeks later;

[0141] MSC-CX group (MSC-CX): 2×10 5 MSC-CX, and 4 weeks later, the hearts of mice with myocardial infarction were subjected to small animal ultrasound detection and Masson's trichrome staining experiments;

[0142] MSC-GY group (MSC-GY): 2×10 5 MSC-GY, and 4 weeks later, the hearts of mice with myocardial infarction were subjected to small animal ultrasound examination and Masson's trichrome staining experiments;

[0143] MSC-CJ group (MSC-CJ): 2×10 5 Four weeks later, the hearts of mice with myocardial infarction were examined by small animal ultrasound and Masson's trichrome staining.

[0144] The results of ultrasound examination of small animals in each group are as follows Fig. 9 The results of Masson's trichrome staining in each group are shown in Fig.10 The results of Masson's trichrome staining were statistically analyzed. Fig.10 As shown in B.

[0145] 2. Experimental Results

[0146] The results of small animal cardiac ultrasound examination ( Fig. 9 ) found that compared with the control group (Ctrl), injection of MSC-GY and MSC-CJ significantly improved the ejection fraction of the heart of myocardial infarction mice ( Fig. 9 A) and short axis shortening rate ( Fig. 9 C), reduced the left ventricular end-systolic diameter ( Fig. 9 B) and left ventricular end-diastolic diameter ( Fig. 9 D), while injection of MSC-CX had little effect on the ejection fraction, fractional shortening, left ventricular end-systolic diameter and left ventricular end-diastolic diameter of the heart of myocardial infarction mice ( Fig. 9 AD). The results of Masson's trichrome staining experiment ( Fig.10 A and B) found that the injection of MSC-GY and MSC-CJ reduced the heart injury area of ​​​​myocardial infarction mice, while the injection of MSC-CX had little effect on the heart injury area of ​​​​myocardial infarction mice. The above results prove that MSC-GY and MSC-CJ have an effective effect on the treatment of myocardial infarction, while MSC-CX has a poor effect on the treatment of myocardial infarction.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method using N-cadherin + / CD168 - A method for screening umbilical cord mesenchymal stem cells subpopulations for effectively treating myocardial infarction, characterized in that: include: Obtaining umbilical cord mesenchymal stem cells; Incubating the umbilical cord mesenchymal stem cells with N-cadherin-PE antibody and CD168-FITC antibody to obtain incubated umbilical cord mesenchymal stem cells; Detection of N-cadherin + / CD168 - The ratio of the umbilical cord mesenchymal stem cell subpopulation in the incubated umbilical cord mesenchymal stem cells, and the effectiveness of the umbilical cord mesenchymal stem cells in treating myocardial infarction is predicted according to the ratio: When the ratio is greater than 25%, it is predicted that the umbilical cord mesenchymal stem cells have an effective effect in treating myocardial infarction.

2. The method according to claim 1, characterized in that The number of the umbilical cord mesenchymal stem cells is (2-5)×10 5 indivual.

3. The method according to claim 1, characterized in that The volume of the N-cadherin-PE antibody is 5 μl to 10 μl, and the volume of the CD168-FITC antibody is 1 μl to 2 μl.

4. The method according to claim 1, characterized in that: The specific process of the incubation is: The umbilical cord mesenchymal stem cells are subjected to a first centrifugation, and then the umbilical cord mesenchymal stem cells after the first centrifugation are subjected to a first washing with a first PBS buffer, and the supernatant is removed after the first washing to obtain an umbilical cord mesenchymal stem cell precipitate; a cell staining buffer, an N-cadherin-PE antibody, and a CD168-FITC antibody are added to the umbilical cord mesenchymal stem cell precipitate for incubation, and after incubation, a second centrifugation is performed and the supernatant is removed, and a second PBS buffer is added to obtain the incubated umbilical cord mesenchymal stem cells.

5. The method according to claim 4, characterized in that The first centrifugation has a rotation speed of 1000 r / min to 1500 r / min and a time of 3 min to 5 min; And / or, the first cleaning is performed 2 to 5 times; And / or, the incubation temperature is 0°C to 4°C and the time is 20min to 30min; And / or, the second centrifugation has a rotation speed of 1000 r / min to 1500 r / min and a time of 3 min to 5 min.

6. The method according to claim 4, characterized in that The volume of the first PBS buffer is 2 mL to 5 mL; and / or, the volume of the cell staining buffer is 100 μl to 200 μl; And / or, the volume of the second PBS buffer is 500 μl to 1000 μl.

7. The method according to claim 1, characterized in that Before the incubation, the umbilical cord mesenchymal stem cells are pretreated, and the specific process of the pretreatment is: Inoculating the umbilical cord mesenchymal stem cells into a culture medium and culturing them for 4 to 5 days, then removing the culture medium; then washing and digesting the umbilical cord mesenchymal stem cells for a second time and then adding the culture medium; and removing the supernatant after a third centrifugation to obtain a cell precipitate; The cell pellet is resuspended to obtain pretreated umbilical cord mesenchymal stem cells.

8. The method according to claim 7, characterized in that The culture medium is a culture medium containing 5 vol% fetal bovine serum, 1 vol% double antibody and 1 vol% mesenchymal stem cell additive; And / or, the third centrifugation has a rotation speed of 1000 r / min to 1500 r / min and a time of 3 min to 5 min.

9. Umbilical cord mesenchymal stem cells screened and obtained by the method according to any one of claims 1 to 8.

10. A stem cell treatment drug for myocardial infarction, characterized in that: Containing the umbilical cord mesenchymal stem cells according to claim 9.

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