CN114085812B - Mesenchymal stem cell population with high expression of CD106 and/or CD142 and reduced expression, and preparation method and application thereof - Google Patents

Mesenchymal stem cell population with high expression of CD106 and/or CD142 and reduced expression, and preparation method and application thereof Download PDF

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CN114085812B
CN114085812B CN202210033348.5A CN202210033348A CN114085812B CN 114085812 B CN114085812 B CN 114085812B CN 202210033348 A CN202210033348 A CN 202210033348A CN 114085812 B CN114085812 B CN 114085812B
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盛宏霞
时兆田
乐宝玉
陈晓颖
侯小强
吉建飞
王凤
李梁
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Bosheng Zhuoyue Biological Science & Technology Beijing Co ltd
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Abstract

The invention provides a mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142, a preparation method and application thereof, and particularly relates to a mesenchymal stem cell population cultured by inflammatory factors (IFN-gamma and/or TNF-alpha), wherein the expression level of CD106 is increased, the expression level of tissue factors (CD 142/TF) is reduced, the expression level of CD274 can be increased, and the expression level of CD80 is unchanged. The mesenchymal stem cell population can reduce the coagulation cascade reaction excited by tissue factors and enhance the immunosuppressive capacity and the cell migration/chemotaxis capacity.

Description

Mesenchymal stem cell population with high expression of CD106 and/or CD142 and reduced expression, and preparation method and application thereof
Technical Field
The invention relates to the field of stem cell culture and biomedicine. In particular to a mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142, a preparation method thereof, a culture and application thereof. After the mesenchymal stem cell population is cultured by the inflammatory factors (IFN-gamma and/or TNF-alpha), the expression level of the immune regulatory factor CD106 is increased, and/or the expression level of the CD142 tissue factor (CD 142/TF) is reduced, and optionally, the expression level of the immune regulatory factor CD274 and the migration/chemotaxis capacity of the mesenchymal stem cell population are increased, but the expression level of the CD80 is unchanged.
Background
Mesenchymal Stem Cells (MSCs) are adult Stem cells that originate in the mesoderm and have a strong self-renewal capacity and a multipotentiality. Mesenchymal stem cells have the ability to promote angiogenesis, promote the regenerative repair of damaged tissues, and relieve various immunological or inflammatory diseases, such as Graft-versus-host disease (GVHD), a disease caused by immune attack of host organs by T lymphocytes in allogenic donor grafts. It can modulate immune activities such as suppression of lymphocyte proliferation, suppression of pro-inflammatory T cell subsets and promotion of immunosuppressive T cell subsets by direct cell contact and/or secretion of soluble factors. The compound has the characteristics of wide sources, easy separation and culture, strong proliferation capacity, low immunogenicity, strong immunoregulation capacity, no ethical problem and the like, and is considered to be a new technology with great potential for curing inflammatory or immune related diseases.
CD106 is involved in the adhesion of lymphocytes, monocytes, and eosinophils and basophils to the vascular endothelium. It regulates T cell, B cell and hematopoietic progenitor cell migration. It has been shown that high expression of CD106 can enhance the immunomodulatory capacity of MSCs and the migration of hematopoietic progenitor cells. Methods for improving the expression of purified CD106 positive cells by flow sorting techniques exist, but have a number of drawbacks: firstly, the sorting antibody has high cost, secondly, the antibody is easy to pollute and the yield is low, which often cannot meet the treatment requirement, and thirdly, the CD106 expressed by the cells can be lost in the in vitro culture and amplification process of the sorted CD106 positive cells.
CD274 (also known as PD-L1, B7H 1) is a ligand for PD-1 (also known as CD 273) and CD 80. CD274 consists of Antigen Presenting Cells (APCs). CD80 is expressed by nasal T cells and is upregulated early in T cell activation, whereas CD274 is expressed late in T cell activation. The interaction of CD274/PD-L1 and PD-1 can transmit inhibitory signals and reduce lymph node CD8+Proliferation of T cells. PD-1 also regulates the Bcl-2 gene, controlling the accumulation of antigen-specific T cells in lymph nodes. Studies have shown that interaction of CD274 (PD-L1)) with CD80 exacerbates the severity of graft versus host disease, while blocking the interaction of CD274 and CD80 in the absence of PD-1 may improve acute GVHD.
Tissue Factor (TF), also known as the cell surface antigen CD142, is a receptor and cofactor for factor vii/viia on the cell surface, initiating the coagulation cascade.
Long-term dysregulation of the immune system in the body can lead to a variety of inflammatory and autoimmune diseases, including Inflammatory Bowel Disease (IBD), graft-versus-host disease (GVHD), type I diabetes, and the like. MSCs can modulate immune activity through direct cell contact and/or secretion of soluble factors, and are considered to be a novel technology with great potential for curing inflammatory or immune-related diseases. However, the decrease in cell quality during in vitro expansion, the low cell viability after in vivo transplantation and the low efficiency of migration to the target site limit the effectiveness of MSC therapy, and the differential expression of tissue factor from mesenchymal stem cells from different sources may trigger an immediate blood-mediated inflammatory response (IBMIR) in the patient population and further increase the risk of thromboembolism.
There have been many studies aimed at improving the immunomodulatory capacity of MSCs, such as flow sorting, various inflammatory factor pretreatment and hypoxic/hypoxic pretreatment. The anoxic/hypoxic treatment has higher requirements on the culture environment and the detection environment, and the flow type separation can bring about the problems of labor cost and pollution. However, few methods for reducing tissue factor of mesenchymal stem cell have been reported.
Mesenchymal stem cells separated by the prior art have strong immunosuppressive capability in vitro, but have nonuniform curative effects in clinical treatment of diseases, the proportion of separated MSCs surface antigens CD274 is less than or equal to 2 percent, the proportion of CD106 positive subgroups is 15-40 percent, and the proportion of MSCs CD142 from different tissue sources is nonuniform, so that the mesenchymal stem cells have potential risks to patients during intravenous injection. Therefore, the current research aims to improve the immunoregulation and migration ability of MSCs to the target site and reduce the expression of tissue factor, and based on this, it is necessary to improve the expression of CD106 and/or CD142 of mesenchymal stem cells and improve the immunoregulation ability of mesenchymal stem cells, which is important for developing safer and more effective MSCs therapy and clinical transformation.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, the present invention is directed to improving the immunomodulatory and target site migration/chemotactic capabilities of MSCs while reducing tissue factor expression, and to producing a more efficient and safer cell product. Thus:
in the first aspect of the present invention, a mesenchymal stem cell population is provided, wherein the expression of the high expression CD106 and/or CD142 of the mesenchymal stem cell population is reduced, the high expression CD106 means that the proportion of CD106 positive subset in the mesenchymal stem cell population is at least 45%, and the reduction of the expression of the CD142 means that the proportion of CD142 positive subset in the mesenchymal stem cell population is reduced by at least 4% after the mesenchymal stem cell population is cultured.
Preferably, the mesenchymal stem cell population is high expressing CD 106;
preferably, the mesenchymal stem cell population has reduced CD142 expression;
preferably, the mesenchymal stem cell population has high expression of CD106 and reduced expression of CD 142.
More preferably, the population of mesenchymal stem cells also highly expresses CD 274; and/or, the mesenchymal stem cell population has no change in the expression level of CD 80.
More preferably, the proportion of CD106 positive subpopulations is at least 45%, and may be any value greater than or equal to 45%, such as at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 81%, 82%, 85%, 86%, 88%, 90%, 92%, 95%, or even higher, and still more preferably, the proportion of CD106 positive subpopulations is at least 80%.
More preferably, the decreased expression level of CD142 in the mesenchymal stem cell population means that the proportion of the CD142 positive subpopulation is decreased by at least 4%, such as at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc., further preferably, the proportion of the CD142 positive subpopulation is decreased by at least 10%, such as at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, etc., and in one embodiment, the proportion of the CD142 positive subpopulation is decreased by more than 20%.
More preferably, the high expression of CD274 means that the percentage of CD274 positive subpopulations is at least 20%, and may be any value greater than or equal to 20%, such as at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, and the like. Further preferably, said high expression of CD274 means that the proportion of CD274 positive subpopulations is at least 60%.
Preferably, the expression level of the mesenchymal stem cell population CD80 is unchanged, and the unchanged expression level of the mesenchymal stem cell population CD80 means that the expression levels of CD80 of the mesenchymal stem cell populations before and after the culture of the mesenchymal stem cell populations have no statistically significant difference.
In the present invention, the expression level and expression have substantially the same meaning and can be expressed by the proportion of the positive subpopulation, for example, high expression of CD106 means that the proportion of the CD106 positive subpopulation is high in the mesenchymal stem cell population, and decreased expression of CD142 means that the proportion of the CD142 positive subpopulation is decreased in the mesenchymal stem cell population, and the like.
Preferably, the mesenchymal stem cell population has enhanced cellular immunosuppressive ability, e.g., inhibition of TNF- α secretion from peripheral blood mononuclear cells, a reduced proportion of Th1 cell population, immunomodulatory factors IL-6 and/or PGE2The amount of secretion increases.
In a specific embodiment, the mesenchymal stem cell population inhibits the secretion of TNF-alpha from peripheral blood mononuclear cells, the proportion of the Th1 cell population is reduced, and the immune regulatory factor isIL-6 and PGE2The amount of secretion increases.
Preferably, the population of mesenchymal stem cells has enhanced cell migration/chemotactic capacity.
Preferably, the population of mesenchymal stem cells is cultured with an inflammatory factor comprising IFN- γ and/or TNF- α.
Preferably, the inflammatory factors are 5-30ng/mL, preferably 5-20 ng/mL, more preferably 8-15 ng/mL, and can be any value between 5-30ng/mL, such as 5.5, 6.0, 7.0, 8.0, 10, 12, 15, 18, 20, 23, 25, 28, 30ng/mL, and the like.
More preferably, the inflammatory factor is IFN- γ.
The mesenchymal stem cell population is high-expressing CD 106;
the mesenchymal stem cell population is high expressing CD 274;
(ii) a decrease in CD142 expression of the population of mesenchymal stem cells; and the combination of (a) and (b),
(ii) no change in CD80 expression of the mesenchymal stem cell population;
said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the mesenchymal stem cell population inhibits TNF-alpha secretion from peripheral blood mononuclear cells, the proportion of the Th1 cell population is reduced, and/or IL-6 and PGE2The secretion amount of (2) is increased.
More preferably, the inflammatory factor is TNF- α.
The mesenchymal stem cell population has high expression of CD106, reduced expression of CD142, and the mesenchymal stem cell population has unchanged expression of CD 80;
said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the mesenchymal stem cell population inhibits TNF-alpha secretion from peripheral blood mononuclear cells, the proportion of the Th1 cell population is reduced, and/or IL-6 and PGE2The secretion amount of (2) is increased.
More preferably, the inflammatory factors are IFN-gamma and TNF-alpha.
The mesenchymal stem cell population is high-expressing CD 106;
(ii) a decrease in CD142 expression of the population of mesenchymal stem cells;
the mesenchymal stem cell population is highly expressing CD274, and,
(ii) the mesenchymal stem cell population has no increase in CD80 expression;
said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the mesenchymal stem cell population inhibits TNF-alpha secretion from peripheral blood mononuclear cells, the proportion of Th1 cell population is reduced, IL-6 and PGE2The secretion amount of (2) is increased.
In a specific embodiment, the inflammatory factor does not comprise an inflammatory factor other than IFN- γ and TNF- α.
In a second aspect of the invention, there is provided a use of an inflammatory factor comprising IFN- γ and/or TNF- α in the culture of a mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD 142.
Preferably, the inflammatory factors are 5-30ng/mL, preferably 5-20 ng/mL, more preferably 8-15 ng/mL, and can be any value between 5-30ng/mL, such as 5.5, 6.0, 7.0, 8.0, 10, 12, 15, 18, 20, 23, 25, 28, 30ng/mL, and the like.
Preferably, the population of mesenchymal stem cells is a population of mesenchymal stem cells according to the first aspect of the invention.
Preferably, the inflammatory factor enhances the immunosuppressive ability and/or cell migration/chemotactic ability of the mesenchymal stem cell population.
More preferably, the inflammatory factor is IFN- γ.
The mesenchymal stem cell population is high-expressing CD 106;
the mesenchymal stem cell population is high expressing CD 274;
(ii) a decrease in CD142 expression of the population of mesenchymal stem cells; and the combination of (a) and (b),
(ii) no change in CD80 expression of the mesenchymal stem cell population;
said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the mesenchymal stem cell population inhibits TNF-alpha secretion from peripheral blood mononuclear cells, the proportion of the Th1 cell population is reduced, and/or IL-6 and PGE2The secretion amount of (2) is increased.
More preferably, the inflammatory factor is TNF- α.
The mesenchymal stem cell population has high expression of CD106, reduced expression of CD142, and no change of expression of CD 80;
said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the mesenchymal stem cell population inhibits TNF-alpha secretion from peripheral blood mononuclear cells, the proportion of the Th1 cell population is reduced, and/or IL-6 and PGE2The secretion amount of (2) is increased.
More preferably, the inflammatory factors are IFN-gamma and TNF-alpha.
The mesenchymal stem cell population is high-expressing CD 106;
the mesenchymal stem cell population is high expressing CD 274;
(ii) a decrease in CD142 expression of the population of mesenchymal stem cells; and the combination of (a) and (b),
the mesenchymal stem cell population has no increase in CD80 expression.
Said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the mesenchymal stem cell population inhibits TNF-alpha secretion from peripheral blood mononuclear cells, the proportion of Th1 cell population is reduced, IL-6 and PGE2The secretion amount of (2) is increased.
In a specific embodiment, the inflammatory factor does not comprise an inflammatory factor other than IFN- γ and TNF- α.
In a third aspect of the invention, there is provided a culture medium comprising a basal medium and an inflammatory factor comprising IFN- γ and/or TNF- α.
Preferably, the inflammatory factors of the culture medium are all 5-30ng/mL, preferably 5-20 ng/mL, more preferably 8-15 ng/mL, and can be any value between 5-30ng/mL, such as 5.5, 6.0, 7.0, 8.0, 10, 12, 15, 18, 20, 23, 25, 28, 30ng/mL, and the like.
More preferably, the inflammatory factor is IFN- γ.
More preferably, the inflammatory factor is TNF- α.
Further preferably, the inflammatory factors are IFN-gamma and TNF-alpha.
In a specific embodiment, the inflammatory factor does not comprise an inflammatory factor other than IFN- γ and TNF- α.
Preferably, the basal medium may be any suitable basal medium for culturing the mesenchymal stem cell population. For example: MSC serum-free medium, DMEM/F12, TL, DMEM, alpha-MEM, F-12, MEM and any combination thereof; more preferably, the basal medium is selected from the group consisting of alpha-MEM, DMEM/F12, TL, DMEM. Further preferably, the basal medium is an MSC serum-free medium.
In a fourth aspect of the present invention, an application of the above culture medium in culturing a mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142 is provided.
Preferably, the population of mesenchymal stem cells is a population of mesenchymal stem cells according to the first aspect of the invention.
More preferably, the inflammatory factor of the medium is IFN- γ.
The mesenchymal stem cell population is high-expressing CD 106;
the mesenchymal stem cell population is high expressing CD 274;
(ii) a decrease in CD142 expression of the population of mesenchymal stem cells; and the combination of (a) and (b),
the mesenchymal stem cell population has no change in CD80 expression.
Said mesenchymal stem cell has increased migration/chemotactic capacity;
preferably, the TNF-alpha secretion amount of the mesenchymal stem cell population is reduced, the proportion of the Th1 cell population is reduced, and IL-6 and PGE are added2The secretion amount of (2) is increased.
More preferably, the inflammatory factor of the medium is TNF- α.
The mesenchymal stem cell population is high in expression of CD106, reduced in expression of CD142, and/or not increased in expression of CD 80.
Preferably, the TNF-alpha secretion amount of the mesenchymal stem cell population is reduced, the proportion of the Th1 cell population is reduced, and IL-6 and PGE are added2The secretion amount of (2) is increased.
The mesenchymal stem cell migration/chemotaxis ability is increased.
More preferably, the inflammatory factors of the culture medium are IFN-gamma and TNF-alpha.
The mesenchymal stem cell population is high in expression of CD106
The mesenchymal stem cell population is high expressing CD 274;
(ii) a decrease in CD142 expression of the population of mesenchymal stem cells; and the combination of (a) and (b),
the mesenchymal stem cell population has no increase in CD80 expression.
The mesenchymal stem cell migration/chemotaxis ability is increased.
Preferably, the TNF-alpha secretion amount of the mesenchymal stem cell population is reduced, the proportion of the Th1 cell population is reduced, and IL-6 and PGE are added2The secretion amount of (2) is increased.
In a specific embodiment, the inflammatory factor does not comprise an inflammatory factor other than IFN- γ and TNF- α.
In a fifth aspect of the present invention, there is provided a method for preparing a mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142, the method comprising culturing the mesenchymal stem cell population with any one of the above-mentioned culture media.
1) Culturing the mesenchymal stem cell population;
2) culturing the mesenchymal stem cell population until the cell fusion degree is 60-70%, and adding inflammatory factors.
Preferably, the thawing recovery step is performed on the mesenchymal stem cell population before the culturing of the mesenchymal stem cell population.
More preferably, the thawing recovery step comprises thawing the cells in a water bath and resuspending the cells in a buffer.
Preferably, the culture is carried out for 16 to 30 hours, more preferably for 18 to 26 hours, and still more preferably for 20 to 24 hours after the addition of the inflammatory factor.
Preferably, the method comprises fractionating the population of mesenchymal stem cells.
More preferably, the ranking comprises establishing a primary cell bank (P2), a master cell bank (P4), and a working cell bank (P5) for the mesenchymal stem cell population.
Further preferably, the grading comprises quality testing of cell banks at various levels.
In a specific embodiment, the P2 generation cells are subjected to quality tests including, but not limited to: one or more of cell morphology, cell number and viability, cell phenotype, differentiation capacity, genetic markers, sterility, mycoplasma, endotoxin, specific human viruses, immunological reactions and cytokine secretion, and qualified as a primary cell bank (P2).
In a specific embodiment, the P4 generation cells are subjected to quality tests including, but not limited to: the cell shape, cell number and survival rate, sterility, mycoplasma and endotoxin, and qualified as the main cell bank (P4).
Preferably, the mesenchymal stem cells of P4 generation are selected to be cultured by the inflammatory factors. More preferably, mesenchymal stem cells of the master cell bank (P4) are selected.
In a specific embodiment, the cells of P5 generation are obtained after culturing with inflammatory factors, and the quality test of the cells of P5 generation is performed, and the quality test includes but is not limited to: cell morphology, cell number and viability, cell phenotype, differentiation capacity, cytogenetics, telomerase, genetic markers, sterility, mycoplasma, endotoxin, specific human viruses, immunological reactions and cytokine secretion, and qualified as a working cell bank (P5).
The grading makes the mesenchymal stem cell population more suitable for clinical use, and the mesenchymal stem cell population has higher safety and more functions.
Preferably, the mesenchymal stem cell population obtained by the preparation method is the mesenchymal stem cell population of the first aspect of the invention.
In a sixth aspect of the present invention, there is provided a culture supernatant prepared from the above culture.
Preferably, the mesenchymal stem cell population and culture supernatant comprise IL-6 and PGE2And/orExosomes of mesenchymal stem cell populations.
In a seventh aspect of the present invention, there is provided a method for producing a cytokine, comprising isolating the cytokine from the culture supernatant.
Preferably, the cytokine comprises IL-6 and/or PGE2。
In an eighth aspect of the present invention, there is provided an application of the above mesenchymal stem cell population, culture supernatant or cytokine, the application comprising:
1) use in research, drug development associated with a CD106 phenotype, a CD274 phenotype, a CD142 phenotype, and/or a CD80 phenotype;
2) use in reducing the coagulation cascade associated with CD 142; and/or the presence of a gas in the gas,
3) use in immunomodulation.
In a ninth aspect of the present invention, an application of the above mesenchymal stem cell population, culture supernatant or cytokine in preparing a composition is provided.
Preferably, the composition is a pharmaceutical composition.
In a tenth aspect of the present invention, there is provided a composition comprising the above mesenchymal stem cell population, culture supernatant, or cytokine.
Preferably, the composition is a pharmaceutical composition.
More preferably, the composition is an injection, a micro-injection, a mucosal patch, an enema, a gel, an aerosol, a drop, and the like.
The pharmaceutical composition optionally comprises pharmaceutical excipients. Further preferably, the adjuvant comprises a pharmaceutically acceptable carrier, diluent and/or buffer, etc.
In some specific embodiments, the carrier is selected from gelatin, chitosan, sodium alginate, collagen, fibrin, polylactic acid, polyurethane, polyethylene oxide, polyethylene glycol, polylactic acid, chitosan acetate, silicate, extracellular matrix, decellularized scaffold, or any combination thereof.
In an eleventh aspect of the invention, there is provided a method of treatment of an immune and/or inflammatory related disease, the method of treatment comprising administering to a subject any of the pharmaceutical compositions described above.
Preferably, the individual is a mammal, such as a human.
Preferably, the above pharmaceutical composition can be used for treating diseases related to immunity and/or inflammation, more preferably, the diseases include blood system diseases, cardiovascular diseases, nervous system diseases, digestive system diseases, reproductive system diseases, urinary system diseases, autoimmune diseases, diseases and injuries in the regenerative medicine/repair medicine field, and the like, for example, Graft Versus Host Disease (GVHD), Ulcerative Colitis (UC), type I diabetes, diabetic foot, spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus, systemic sclerosis, crohn's disease, stroke, cirrhosis, hematopoietic reconstitution, and the like.
The mesenchymal stem cell population is derived from umbilical cord blood, bone marrow, fat, synovial membrane, bone, muscle, lung, liver, pancreas, amniotic fluid, and the like.
Description of related terms of the invention
Mesenchymal stem cells (mesnschel stem cells, MSC): is a pluripotent stem cell that has all of the commonality of a stem cell, namely, self-renewal and pluripotency.
Umbilical cord Mesenchymal Stem Cells (Umbilical code Mesenchyl Stem Cells, UC-MSCs): stem cells isolated from umbilical cord tissue of a newborn. The tissue has the name of being capable of differentiating into interstitial tissues, has sub-totipotent differentiation potential, and can be induced to differentiate into various tissue cells under specific in vivo and in vitro environments. Umbilical cord mesenchymal stem cells have the commonality of stem cells, i.e., the ability to self-renew, differentiate in multiple directions, and home, and are referred to by scientists as "powerful progenitor cells".
Graft Versus Host Disease (GVHD): since the T lymphocytes in the allodonor graft after transplantation are stimulated by a series of "cytokine storms" initiated by the recipient, their immune response to the recipient antigen is enhanced, targeting the recipient target cells to initiate cytotoxic attack, with skin, liver and gut being the primary targets. The incidence rate of acute graft-versus-host disease is 30% -45%, and the incidence rate of chronic patients is lower than that of acute patients.
Human Peripheral Blood Mononuclear Cells (PBMC): a heterogeneous population of mononuclear blood cells, including macrophages, dendritic cells, monocytes and lymphocytes.
Immediate blood-mediated inflammatory response (IBMIR): the graft causes the activation of the blood coagulation system of the graft recipient and the activation of the complement system secondarily, so that cascade reaction occurs, and the activation, aggregation and neutrophil adhesion of blood platelets are caused.
Tissue Factor (TF), cell surface antigen CD142 (CD 142 antigen), is a receptor and cofactor for factor VII/VIIa on the cell surface, and is a promoter in the extrinsic coagulation process. TF does not exist in circulation or does not contact with circulating blood under normal conditions so as to ensure smooth blood flow and maintain normal blood circulation function, only when the integrity of a blood vessel wall is damaged, a large amount of endothelial cells are expressed, TF is exposed to the circulating blood, and under the condition of the existence of calcium and phospholipid, Coagulation cascade reaction (Coagulation cascade) is started by combining with Coagulation factor VII/VIIa to play a role in hemostasis.
Vascular Cell Adhesion Molecule-1 (VCAM-1), also known as CD106, is an Adhesion Molecule that belongs to the immunoglobulin family. Present on activated endothelial cells, tissue macrophages, dendritic cells and bone marrow fibroblasts. It is involved in the adhesion of lymphocytes, monocytes and eosinophils to the active endothelium. It regulates T cell, B cell and hematopoietic progenitor cell migration. Research shows that the high expression of CD106 can improve the immunoregulation capability of MSCs.
Programmed cell death ligand (PD-L1), also known as CD274, encodes an immunosuppressive receptor ligand and is expressed in hematopoietic and non-hematopoietic cells (e.g., T-and B-cells) as well as various types of tumor cells. Through interaction with its receptor, inhibits the activation of living T cells and the production of cytokines. This interaction prevents autoimmunity by maintaining a balance of immune responses during infection or inflammation of normal tissues.
Cluster of differentiation 80 (CD 80), also known as B7-1, belongs to the immunoglobulin superfamily, is a costimulatory factor for CD86 to activate T lymphocytes, and plays an important role in autoimmune monitoring, humoral immune response, and transplantation response. Expressed on activated B lymphocytes, T lymphocytes, macrophages, peripheral blood monocytes and dendritic cells, but not on non-activated B lymphocytes, erythrocytes, granulocytes and monocytes.
The invention has the advantages of
1. According to the invention, the MSC is treated by IFN-gamma or/and TNF-alpha, so that the proportion of the CD106 positive subgroup is increased from 15-40% to 80-90%, and the immunoregulation capability and cell migration capability of the mesenchymal stem cell population are improved. In a preferred embodiment, the combination of IFN- γ and TNF- α is synergistic in increasing the expression of CD 106.
2. The invention utilizes the inflammatory factors to culture the mesenchymal stem cell population for the first time to reduce the phenotype of the CD142, and in a preferred scheme, the combination of IFN-gamma and TNF-alpha has a synergistic effect on reducing the expression of the CD 142. The proportion of the CD142 positive subgroup is reduced by 20 percent, the blood coagulation cascade reaction caused by high CD142 when the mesenchymal stem cell population is used is avoided, and the risks of IBMIR, thromboembolism and the like are reduced.
3. In the preferable scheme, the combination of IFN-gamma and TNF-alpha has a synergistic effect on improving the expression of CD274, can be improved to 85 percent, and can further inhibit the adverse effect of CD 142; and the proportion of CD80 is not increased while CD274 is highly expressed, so that the potential danger of exacerbating the condition of the GVHD patient is avoided.
4. The invention uses IFN-gamma or/and TNF-alpha to treat the mesenchymal stem cells, so that the migration/chemotaxis capability of the cells is increased, and the quantity of the MSCs chemotactic/migrated to a target organ is increased during the treatment of diseases, so that the MSC can better play the role of immunoregulation.
5. The invention utilizes one or two inflammatory factors to culture the mesenchymal stem cell population, so that the mesenchymal stem cell population has various performances, the method is simple to operate, only ordinary cell culture operation is needed, and the problems of labor cost and pollution caused by flow type sorting are avoided.
Drawings
FIG. 1 shows the results of the change of positive subpopulations of CD274, CD80 and CD106 after the umbilical cord mesenchymal stem cell population is treated with IFN-gamma or/and TNF-alpha;
FIG. 2 shows the result of the change of CD142 positive subset after IFN-gamma or/and TNF-alpha treatment of the mesenchymal stem cell population; (ii) a
FIG. 3 shows the results of the changes in the phenotype of CD274, CD80 and CD106 cells before and after cryopreservation of hUC-MSCs after IFN-. gamma.or/and TNF-. alpha.treatment;
FIG. 4 shows the results of the change in CD142 positive subset after cryopreservation of hUC-MSCs after IFN- γ or/and TNF- α treatment;
FIG. 5 shows the results of the change of positive subsets of CD274, CD80 and CD106 after IFN-gamma or/and TNF-alpha treatment of hUC-MSCs cultured in different culture media;
FIG. 6 shows the results of the change of CD142 positive subset after IFN-gamma or/and TNF-alpha treatment of hUC-MSC cultured in different culture media;
FIG. 7 shows the results of the change in CD274, CD80 and CD106 positive subsets after treatment of hUC-MSC cells with different concentrations of TNF- α and IFN- γ;
FIG. 8 shows the results of CD142 changes after treatment of hUC-MSC cells with different concentrations of TNF- α and IFN- γ;
FIG. 9 shows the change of immunomodulatory capacity of IFN-. gamma.or/and TNF-. alpha.treated hUC-MSCs after co-culture with PBMCs, wherein (a) different groups of TNF-. alpha.factor secretion; (b) inhibition rate of different groups of hUC-MSC to PBMCs; FIG. c Th1 (IFN-γ +) Cell proportion evaluation results;
FIG. 10 shows the secretion of immunomodulatory factors after treatment of hUC-MSC with inflammatory factors, wherein (a) is the secretion of IL-6; (b) is shown as PGE2Is divided intoThe amount of the feed is secreted;
FIG. 11 is a graph of the effect of IFN-. gamma.or/and TNF-. alpha.treatment on hUC-MSC migration and chemotaxis, wherein (a) is the 0h, 6h cell scratch results; (b) cell mobility was 0h, 6 h; (c) (ii) hUC-MSC cell chemotaxis results for different concentrations of IFN-gamma and TNF-alpha treatment; (d) chemotaxis results for hUC-MSC cells treated with IFN-gamma or/and TNF-alpha at 10 ng/mL.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
In each of the examples described below, the apparatus and reagents were obtained from several companies indicated below
The apparatus used in this description is as follows:
device name Manufacturer of the product Model number
Biological safety cabinet Thermo 1384
Carbon dioxide incubator Thermo HERA cell 150i
Centrifugal machine Thermo ST16R
Cell counter Nexcelom K2
Inverted microscope Mingmei tablet MI11
Automatic plate washing machine Thermo Wellwash
Microplate constant temperature incubator MIULAB ST70-2
Multifunctional enzyme mark instrument Thermo 3020
Flow cytometer BD BD FACSVerse
The reagents used in this specification are as follows:
TNF-alpha (PeproTech 300-01A-50), IFN-gamma (PeproTech AF-300-02-20), Mesenchymal Stem Cell (MSC) serum-Free Medium (Beijing three beneficial science and technology development Co., Ltd. 120408), AMMS Medium Steriled Filter (Kyoho)Sea-derived organism AS 09-2), DMEM/F12 basal medium (Proxel PM 150310), TrypLE Express Enzyme (GIBCO 12604-, Cyto-FastTM Fix/PermBuffer Set（BioLegend 426803）、Cell Staining Buffer（BioLegend 420201）、Viability Stain 510（BD 564406）、CellTrace™ Violet Cell Proliferation Kit（Invitrogen™ C34557）、FITC Anti-Human CD3（BioLegend 300406）、7-AAD Viability Staining Solution（BioLegend 420403）、Human TNF-alpha Quantikine ELISA Kit（R&D STA00D）、Prostaglandin E2 Parameter Assay Kit（R&D SKGE004B）、Human IL-6 Quantikine ELISA Kit Summary（R&D S6050), FITC Mouse anti-human CD274 (BD Pharmingen 7125831), APC anti-human CD80 (BioLegen 305219), APC anti-human CD106 (BioLegen 305809), APC anti-human CD142 (BioLegen 365205), DAPI Solution 1.0 mg (BD Pharmingen 564907), 4% histocyte fixed Solution (Solarbio P1110-100 ml), ammonium oxalate crystal violet staining Solution (crystal violet staining Solution) (1%) (Solarbio G1062-10 ml)
Example 1 Process for establishing three cell banks, sufficient quantities of each cell bank were produced
Isolating primary cells of the human umbilical cord mesenchymal stem cell population under the condition of meeting GMP according to the prior art (for example, CN 103266081B) until the cells of the P2 generation are qualified and become a primary cell bank; thawing the cells of the P2 generation to grow to P4 generation, and obtaining a main cell bank after passing inspection; thawing and recovering the P4 generation cells, inoculating according to a certain concentration, treating the cells for 22 +/-2 h by using inflammatory factors, and then harvesting the P5 generation cells, wherein the cells are a working cell bank after passing inspection.
Wherein the P2 primary cell bank assay includes but is not limited to: one or more of cell morphology, cell number and viability, cell phenotype, differentiation capacity, genetic markers, sterility, mycoplasma, endotoxin, specific human viruses, immunological reactions, and cytokine secretion;
the P4 master cell bank test includes, but is not limited to, the following items: one or more of cell morphology, cell number and viability, sterility, mycoplasma and endotoxin;
p5 work cell library test items include but are not limited to: cell morphology, cell number and viability, cell phenotype, differentiation capacity, cytogenetics, telomerase, genetic markers, sterility, mycoplasma, endotoxin, specific human viruses, immunological reactions, and cytokine secretion. The cells with qualified indexes are the clinical grade stem cells.
Example 2 Change in phenotypes of umbilical cord mesenchymal stem cell populations treated with inflammatory factors CD106, CD274, CD80 and CD142
The culture method comprises the following steps:
1) thawing and recovering: thawing a P4 main library cell bank in water bath at 37-40 ℃, re-suspending the cell suspension in a buffer solution (the buffer solution can be PBS, normal saline and normal saline containing 1% human serum albumin), wherein the re-suspending proportion is at least 2 times of that of the buffer solution, 300-500 Xg, centrifuging for 8-10 min, discarding the supernatant, and adding three beneficial culture media;
2) culturing: according to the ratio of 5000-6000 cells/cm2Inoculating into T75 cell culture flask, and placing the flask at 37 deg.C and 5% CO2Culturing in an incubator;
3) adding inflammatory factors to continue culturing: after 48 +/-2 h, three beneficial MSC serum-free culture media (grouping information is shown in table 1) containing IFN-gamma or/and TNF-alpha with the concentration of 10 ng/mL are replaced, after 22 +/-2 h, the cells are respectively harvested, and 2 multiplied by 10 is taken6Individual cells were used for flow cytometry analysis. The cells examined were washed three times with PBS, resuspended at 200. mu.L, and rootedThe antibody was added according to the instructions for antibody use and incubated at 4 ℃ for 20 min in the absence of light. After incubation was complete, PBS was washed three times, 300. mu.L of DAPI (0.1. mu.g/mL) was resuspended and the cells were examined on a flow cytometer.
Table 1: list of inflammatory factor addition for different groups
1 2 3 4（NTC)
TNF-α + + - -
IFN-γ + - + -
Table 2: results of changes in CD106, CD274, CD80 and CD142 phenotypes after 24h treatment of hUC-MSC with inflammatory factors
Figure 411877DEST_PATH_IMAGE001
As shown in figures 1 and 2 and table 2,
for phenotype CD106, single IFN-gamma can obviously improve the expression level of CD106, while the TNF-alpha group does not obviously improve the ratio, but has the effect of synergistically improving the expression level of CD106 after the two inflammatory factors are combined.
For phenotype CD274, single TNF-alpha or IFN-gamma can improve the expression level of CD274, and the single IFN-gamma can obviously improve the expression level of CD274, and compared with a control group, the proportion of CD274 positive subgroups is increased by about 60 times; the increase of the expression level of CD274 by the combination of the two inflammatory factors is far higher than the sum of the effects of the single inflammatory factors, which indicates that the combination of the two inflammatory factors has a synergistic effect on the increase of the expression level of CD 274.
For the CD142 phenotype, a single IFN-gamma inflammatory factor group has a certain effect of reducing the CD142 phenotype, while the combined inflammatory factor group has the effect of obviously reducing the CD142 by reducing the CD142 phenotype by about 20 percent compared with a control group, and the two inflammatory factors also have synergistic effect on reducing the expression quantity of the CD 142.
There was no significant change in the CD80 phenotype, either in single inflammatory factor cultures or in combination.
Example 3: change of cell phenotype before and after cryopreservation of hUC-MSC treated by inflammatory factor
The culture method comprises the following steps: cells were seeded, treated and harvested according to the method of example 2. Collecting the harvested cell pellet at 2X 106One cell was analyzed by flow cytometry, and the remaining cells were cryopreserved. The phenotype was detected by resuscitation after two months of frozen storage in liquid nitrogen.
As a result:
table 3: detection result of cell phenotype after cryopreservation recovery
Figure DEST_PATH_IMAGE002
As shown in figures 3, 4 and table 3, there was no significant change in CD106, CD274, CD80, and CD142 phenotypes before and after cryopreservation, indicating that the various phenotypes were stably expressed after inflammatory factor treatment.
Example 4: changes in CD106, CD274, CD80 and CD142 phenotypes of hUC-MSCs cultured in different media following treatment with inflammatory factors
The culture method comprises the following steps: recovering the cells of the P4 main cell bank at 37-40 ℃, wherein the cell density is 5000-6000 cells/cm2In a culture bottle inoculated with T25 cells, AMMS MSC Medium bone Free Medium Steriled (TL) and DMEM/F12 +10% FBS (DF 12) were used for culturing, after 48 + -2 h, the culture media (TL and DF 12) containing the inflammatory factors shown in Table 1 were respectively replaced, and after 22 + -2 h, the cells were respectively harvested.
As a result:
table 4: effect of different media on cell phenotype
Figure 17433DEST_PATH_IMAGE003
As shown in fig. 5 and 6 and tables 2 and 4, although the expression level of the cell phenotype was different in the different media, the influence tendency was the same as in example 2, and the influence of the inflammatory factor on the various cell phenotypes was the same even in the medium containing DF12+10% FBS in serum.
For phenotype CD106, single TNF-alpha or IFN-gamma can obviously improve the expression level of CD106, and after the two inflammatory factors are combined, the expression level of CD106 can be synergistically improved.
For phenotype CD274, single TNF-alpha or IFN-gamma can improve the expression level of CD274, and single IFN-gamma can obviously improve the expression level of CD 274; the effect of the combination of the two inflammatory factors on the increase of the expression level of the CD274 is far higher than the sum of the effects of the single inflammatory factors, which indicates that the combination of the two inflammatory factors has a synergistic effect on the increase of the expression level of the CD 274.
There was no significant change in the CD80 phenotype, either in single inflammatory factor cultures or in combination.
For the CD142 phenotype, the combination of inflammatory factors may reduce the proportion of CD142 positive subpopulations, but when the medium is TL, TNF-alpha alone may also reduce the proportion of CD 142.
In summary, the double-addition group resulted in an increase in the ratio of CD106 to CD274 and a constant ratio of CD80 and a decrease in the ratio of CD142, regardless of the medium. Thus, the inflammatory factors of the present invention can be adapted to a variety of different media to achieve the desired effect on cell phenotype.
Example 5: changes in CD106, CD274, CD80 and CD142 phenotypes following treatment with different concentrations of inflammatory factors
The culture method comprises the following steps: recovering the cells of the P4 main cell bank at 37-40 ℃, wherein the cell density is 5000-6000 cells/cm2Inoculating into T25 cell culture bottle, culturing with three beneficial (Sanly) culture media, treating hUC-MSC cells with TNF-alpha and IFN-gamma of 5, 10, 20, 30ng/mL after 48 + -2 hr, respectively, harvesting cells after 22 + -2 hr, collecting 2 × 10 cell precipitate6Individual cells were analyzed by flow cytometry.
As a result:
table 5: results of cell phenotype changes after different concentrations of inflammatory factor treatment of hUC-MSC
Figure DEST_PATH_IMAGE004
As shown in figures 7 and 8 and table 5,
for phenotype CD106, concentrations of inflammatory factors ranging from 5-30ng/mL all had a synergistic effect in increasing CD106 expression;
for phenotype CD274, as the concentration of inflammatory factors increases, the synergistic effect of both on CD274 expression also increases;
for phenotype CD80, there was no significant change in the expression of CD80 as the concentration of inflammatory factors increased;
for phenotype CD142, the proportion of CD142 shows a trend of decreasing and then increasing with the increase of the concentration of inflammatory factors after the combined treatment, but generally, the proportion of CD142 positive is decreased in the range of 5-30ng/mL, and particularly, the proportion is the lowest at 10 ng/mL.
Example 6: effect of inflammatory factor treatment on cellular immunosuppressive Capacity
The method comprises the following steps: inoculation, treatment and harvesting were performed according to the method of example 2. The harvested cells were resuspended and seeded in 6-well plates at 1X 10 per well6Culturing the cells for 24h, collecting supernatant, and detecting IL-6 and PGE2The amount of secretion. After the cells are attached to the wall, mitomycin C with the final concentration of 20 mug/mL is added for treatment for 2 h, the washing is carried out for 3 times, and RPMI 1640 (10% FBS) is added for continuous culture. PBMC and MSC were co-cultured and PBMC cell suspension was added to wells not inoculated with MSC as a positive control. CD3 monoclonal antibody with the final concentration of 5 mu g/mL and CD28 monoclonal antibody with the final concentration of 1 mu g/mL are respectively added into the wells of the experimental group and the positive control group. Cells were incubated at 37 ℃ with 5% CO2After culturing for 3-4 days under the condition, analyzing the proportion of Th1 cell population by using a flow cytometer.
As a result:
table 6: effect of immune regulatory factors after inflammatory factor treatment
Figure 734853DEST_PATH_IMAGE005
Note:Pthe value is the significance analysis (t test) of the inflammatory factor treatment group and the control group; in the tablePA value less than 0.05 indicates significant difference; a value of less than 0.01 indicates that the difference is extremely significant.
As can be seen from figures 9 and 10 and table 6,
as for the amount of TNF-alpha secreted from peripheral blood mononuclear cells, either IFN-gamma alone or TNF-alpha alone or in combination, has the effect of reducing the amount of TNF-alpha secreted from peripheral blood mononuclear cells. For example: the secretion amount of the PBMCs TNF-alpha positive control is 2510 pg/mL, and the reduction of the secretion amount of TNF-alpha of peripheral blood mononuclear cells after the combined treatment of the inflammatory factors can be reduced by 225.1 pg/mL.
For the proportion of Th1 cell population, whether IFN-gamma alone or TNF-alpha alone or in combination, the effect of reducing the proportion of Th1 cell population was observed.
As for the secretion of IL-6, the secretion of IFN-gamma, TNF-alpha or the combination thereof can be increased.
For the immunomodulatory factor PGE2Whether IFN-. gamma.alone or TNF-. alpha.alone or in combination, has the effect of increasing the secretion of IFN-. gamma.s.
The results show that the immunosuppressive capability of the hUC-MSCs after the treatment of the inflammatory factors is enhanced.
Example 7: effect on cell migration after treatment with inflammatory factors
The method comprises the following steps: recovering the cells of the P4 main cell bank at 37-40 ℃ according to 6000 cells/cm2 Culturing with Sanly (Tri-advantageous mesenchymal stem cell serum-free medium) in 6-well plate, and placing the culture flask at 37 deg.C and 5% CO2After 48 h, MSC serum-free medium containing TNF-alpha or IFN-gamma at a concentration of 10 ng/mL and TNF-alpha and IFN-gamma at 5, 10, 20, 30ng/mL was replaced and inflammatory factor treatment was carried out for 22 + -2 h.
Cell scratching: in the test, 10 ng/mL TNF-alpha and/or IFN-gamma is added into mitomycin C with the final concentration of 20 mug/mL for treatment for 2 hours, a gun head is used for scratching, PBS is used for washing for 3 times, then the culture is continued by changing into alpha-MEM basal medium, the scratched area is photographed and observed under a microscope, the photographed position is marked, and 0, 4 and 6 hour scratched pictures are photographed and stored.
Cell chemotaxis: assay TNF-. alpha.and IFN-. gamma.were resuspended at 10 ng/mL and TNF-. alpha.and IFN-. gamma.at 5, 10, 20, 30ng/mL using alpha-MEM (2% FBS) adjusted to a cell density of 5X 105Perml, 200. mu.L of cell suspension was placed in the upper and lower chambers of alpha-MEM (30% FBS) in a total volume of 700. mu.L, and the cells were incubated at 37 ℃ and 5% CO2 After 24h of culture in an incubator, washing with PBS for 3 times, fixing with 4% paraformaldehyde for 30 min, washing with PBS for 5 times, staining with 0.1% crystal violet for 15 min, washing with PBS for 3 times, and collecting 20 × pictures.
As a result:
table 7: cell migration area list
Figure DEST_PATH_IMAGE006
TABLE 8 details of cell migration area after combination treatment with inflammatory factors at different concentrations
Figure 317013DEST_PATH_IMAGE007
Note:Pthe significance analysis (t test) of the inflammatory factor treatment group and the control group with the same time point is carried out; in the tablePA value less than 0.05 indicates significant difference; a value of less than 0.01 indicates that the difference is extremely significant.
As shown in fig. 11 and table 7, the cell migration ability was increased after the treatment with inflammatory factors (fig. 11 a and b); the chemotactic ability of the combination group decreased with increasing concentration, but was higher than that of the control group (FIG. 11 c), indicating that the inflammatory factor concentration was within a certain range; the chemotactic capacity of the group alone was increased compared to the control (FIG. 11 d), and that of the IFN-. gamma.group was higher than that of the TNF-. alpha.and combination groups. As shown in table 8, when the hUC-MSC was treated with different concentrations of the inflammatory factors, the mobility increased with increasing concentration at 4h, and decreased with increasing concentration at 6 h. It was shown that either single plus or double plus treatment at different concentrations resulted in increased hUC-MSC migration, but inflammatory factor concentrations required a suitable range for the cells to function better.
As described above, the cell migration and chemotactic capacity can be increased by single-addition or combined treatment.
Example 8: preparation of composition comprising mesenchymal stem cell population
The functionally enhanced mesenchymal stem cell population composition of the present invention may comprise:
(1) the mesenchymal stem cell population treated by the inflammatory factors has a certain concentration (2 multiplied by 10)5cell/mL ~1×106cell/mL) in physiological saline containing 1% human blood protein for intravenous return infusion;
(2) the mesenchymal stem cell population treated by the inflammatory factors has a certain concentration (2 multiplied by 10)5cell/mL ~1×106cell/mL) in a container containing a certain proportion of propylene glycol, trehalose and humanThe blood albumin + compound electrolyte injection is used for intravenous return; alternatively, the first and second electrodes may be,
(3) after being treated by inflammatory factors, the supernatant secreted by the mesenchymal stem cell group is prepared into a smearing preparation or a spraying agent and the like together with gelatin, chitosan, sodium alginate, collagen, fibrin, polylactic acid, polyurethane, polyethylene oxide, polyethylene glycol, polylactic acid-methanol acetic acid, silicate, extracellular matrix, acellular scaffolds and the like after being subjected to an exosome extraction process.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims (8)

1. The application of an inflammatory factor in culturing a mesenchymal stem cell population to obtain the mesenchymal stem cell population with high expression of CD106 and reduced expression of CD142 is characterized in that the inflammatory factor comprises IFN-gamma and TNF-alpha, the expression of the mesenchymal stem cell population after being cultured by the inflammatory factor is reduced in high expression of CD106 and CD142, the high expression of CD106 means that the proportion of CD106 positive subset in the mesenchymal stem cell population after being cultured is at least 45%, and the reduction of the expression of CD142 means that the proportion of CD142 positive subset in the mesenchymal stem cell population after being cultured is reduced by at least 4%.
2. The use of claim 1, wherein the inflammatory factor is present in the culture medium at a concentration of 5-30 ng/mL.
3. The use according to any one of claims 1 to 2, wherein the inflammatory factor enhances the immunosuppressive ability and/or cell migration/chemotactic ability of the mesenchymal stem cell population.
4. The use of any one of claims 1-2, wherein the mesenchymal stem cell population has no change in the amount of highly expressed CD274 and/or CD80 expression following culture.
5. A preparation method of a mesenchymal stem cell population with high expression of CD106 and reduced expression of CD142 is characterized in that the method comprises the step of culturing the mesenchymal stem cell population by using an inflammation factor or a culture medium containing the inflammation factor, wherein the inflammation factor comprises IFN-gamma and TNF-alpha, the expression of the high expression CD106 and the expression of the CD142 of the mesenchymal stem cell population are reduced after the mesenchymal stem cell population is cultured by the inflammation factor, the high expression CD106 means that the proportion of CD106 positive subset in the mesenchymal stem cell population after the culture is at least 45%, and the expression of the CD142 is reduced means that the proportion of CD142 positive subset in the mesenchymal stem cell population after the culture is at least 4%.
6. The method of claim 5, wherein the inflammatory factor is present in the culture medium at a concentration of 5-30 ng/mL.
7. The method of any one of claims 5-6, wherein the inflammatory factor enhances the immunosuppressive ability and/or cell migration/chemotactic ability of the mesenchymal stem cell population.
8. The method of any one of claims 5-6, wherein the mesenchymal stem cell population has no change in the expression level of highly expressed CD274 and/or CD80 after culture.
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