CD83 + and CD83 +PD-l1 + mesenchymal stem cells, method for preparing same, and use thereof

Abstract

Provided are use of a CD83+ mesenchymal stem cell, a CD83+ overexpressing mesenchymal stem cell or a CD83+PD-L1+ mesenchymal stem cell in preparing a medicament for preventing / treating an autoimmune disease and / or inflammatory disease, and a method for preparing the CD83+ mesenchymal stem cell, the CD83+ overexpressing mesenchymal stem cell or the CD83+PD-L1+ mesenchymal stem cell in the described use. The CD83+ mesenchymal stem cell, the CD83+ overexpressing mesenchymal stem cell, or the CD83+PD-L1+ mesenchymal stem cell can increase the secretion of anti-inflammatory factors and / or immunosuppressive factors, and treat autoimmune diseases such as rheumatoid arthritis and inflammatory diseases such as sepsis. According to the induction process flow of the anti-inflammatory CD83+PD-L1+ mesenchymal stem cell, the detection of the positive expression rate of CD83 and PD-L1 receptors may help the development of kits for evaluating a treatment with the anti-inflammatory CD83+PD-L1+ mesenchymal stem cell.

Description

CD83 + 、CD83 + PD-L1 + Mesenchymal stem cells and their preparation method and application

[0001] This application is filed with the China Patent Office on November 21, 2023, with application number 202311550534.7 and the invention name “CD83 + 、CD83 + PD-L1 + The entire contents of the Chinese patent application entitled "Mesenchymal stem cells, preparation method and application thereof" are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine and specifically relates to an immunosuppressive or anti-inflammatory function-enabling CD83 + Mesenchymal stem cells and CD83 with enhanced immunosuppressive or anti-inflammatory functions + PD-L1 + Mesenchymal stem cells, as well as in vitro screening or gene modification overexpression kits, immunosuppression or anti-inflammatory induction kits, quality assessment methods and applications. Background Art

[0003] With the advancement of stem cell therapy research, mesenchymal stem cells (MSCs) have been recognized as an ideal tool for treating tissue-damaging diseases and immune- or inflammatory-related diseases. MSCs are multipotent stem cells with the potential for self-renewal and multilineage differentiation, widely distributed in adult tissues such as bone marrow, adipose tissue, dental pulp, placenta, and umbilical cord. Numerous studies have demonstrated that MSCs not only possess robust self-renewal capacity and the potential for multilineage differentiation into mesoderm, ectoderm, and endoderm, but also possess excellent immunomodulatory and inflammatory properties. They regulate the proliferation, differentiation, secretion, and polarization of immune cells such as T cells, NK cells, and macrophages, thereby effectively modulating imbalances in the immune and inflammatory response. Furthermore, they exhibit multiple biological properties, such as effectively inhibiting apoptosis of cells in situ within damaged tissues, promoting angiogenesis, and activating stem cells in situ, contributing to the regeneration and repair of damaged tissues. Therefore, MSC transplantation not only restores a patient's imbalanced immune or inflammatory response but also possesses tissue regeneration and repair capabilities. These characteristics make MSC transplantation a promising strategy for the treatment of injury, inflammation, and immune-related diseases.

[0004] It has been confirmed that, in addition to directly participating in the regeneration and repair of damaged tissues through differentiation and secretion of cell growth factors, MSCs can also regulate the imbalance of the body's inflammatory / immune response, repair the body's immune homeostasis, avoid secondary inflammatory damage to target organs, improve the body's tissue repair microenvironment, and promote the regeneration and repair of damaged tissues. Given that MSCs transplantation can treat inflammatory and immune-related diseases, it can not only fundamentally restore the imbalance of the body's inflammatory / immune response, but also promote the regeneration and repair of damaged tissues. Therefore, MSCs transplantation is considered to be an ideal strategy with great potential for treating injury, inflammation, and immune-related diseases. However, with the increase in the amount of clinical data on MSCs transplantation for the treatment of inflammatory / immune-related diseases, it has been found that there are huge individual differences in the clinical efficacy of MSCs transplantation for the treatment of inflammatory / immune-related diseases. Even if some patients with the same disease are cured, some patients are ineffective or even have the opposite results.

[0005] A deeper understanding of the characteristics of MSCs and their inflammatory / immunomodulatory mechanisms has revealed that MSCs naturally lack tissue repair and inflammatory regulatory activities, and their tissue repair and immunomodulatory functions are dependent on microenvironmental stimulation. Regarding tissue repair, MSCs in their resting state lack tissue repair capacity, and their acquisition of this capacity requires stimulation with growth factors such as TGF-β, BMP, HGF, and PDGF. Different stimuli can induce distinct differentiation pathways and growth factor secretion profiles in MSCs, thereby affecting their tissue repair function and resulting in varying regenerative outcomes. Similarly, the inflammatory / immunomodulatory functions of MSCs are not constitutive. In other words, MSCs naturally have low inflammatory / immunomodulatory capabilities, and their acquisition depends on stimulation from the inflammatory / immune microenvironment and is regulated by numerous factors. For example, MSCs require stimulation with inflammatory factors such as IFN-γ, TNF-α, and IL-1β to undergo immunosuppressive or anti-inflammatory reprogramming, subsequently expressing anti-inflammatory mediators or receptors such as PD-L1, IDO, and IL-10, in order to exert their immunosuppressive or anti-inflammatory effects. Furthermore, although MSCs are currently believed to possess potent immunosuppressive or anti-inflammatory functions, their regulation of host inflammation / immunity exhibits a bidirectional nature, capable of both activating and promoting immunity and suppressing or suppressing it. This dual nature of MSCs in both inflammation and immunity significantly impacts their clinical efficacy in treating inflammatory / immune diseases. TGF-β1 and the TLR4 agonist LPS have been shown to induce MSCs to reprogram toward either an immunoactivating or proinflammatory phenotype, exerting proinflammatory effects. TLR3 agonists or inflammatory factors (such as IFN-γ, IL-1β, and TNF-α) induce MSCs to reprogram toward either an anti-inflammatory or immunosuppressive phenotype, exerting anti-inflammatory effects. However, due to differences in etiology and disease course, patients with numerous inflammatory / immune-related diseases often experience the coexistence of factors or mediators in their microenvironment that can both induce and suppress MSCs' pro-inflammatory and anti-inflammatory properties, leading to the coexistence of pro- and anti-inflammatory MSCs. Alternatively, some patients lack anti-inflammatory factors, hindering the anti-inflammatory reprogramming of MSCs. The above reasons may be the main reasons for the significant individual differences in treatment resistance and clinical efficacy of MSC transplantation for inflammatory / immune-related diseases. Therefore, how to empower and maintain the anti-inflammatory or immunosuppressive function of MSCs is a key technical link in improving the clinical efficacy of MSC transplantation for inflammatory / immune-related diseases.

[0006] A large number of studies have confirmed that MSCs are a heterogeneous group of multipotent stem cells with differences in multifunctional subpopulations and tissue origins. Wang Z et al. used scRNA-seq to compare the differences in MSCs from adipose, bone marrow, and umbilical cord, and found 7 tissue-specific and 5 functionally conservative MSCs subpopulations. In comparison, umbilical cord MSCs (hUC-MSCs) have stronger inflammatory regulation potential. Zhang S et al. found two functional subpopulations with different inflammatory regulation and tissue differentiation functions in primary MSCs. However, although there have been studies on the heterogeneity of MSCs, they have only simply compared their differences in tissue origin and lack characteristic information of functional groups. In addition, the heterogeneity data obtained from static MSCs are difficult to represent the spatiotemporal empowerment programming information of the inflammatory regulation subpopulation of MSCs during the process of inflammatory diseases.

[0007] Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the main purpose of the present invention is to provide a CD83 cell line that is easy to be enabled with immunosuppressive or anti-inflammatory function. + Mesenchymal stem cells and CD83 with enhanced immunosuppressive or anti-inflammatory functions + PD-L1 + Mesenchymal stem cells and their preparation method; the present invention also provides the immunosuppressive or anti-inflammatory function easily enabled CD83 + Mesenchymal stem cells and CD83 with enhanced immunosuppressive or anti-inflammatory functions + PD-L1 + Applications of mesenchymal stem cells. Specifically:

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] First aspect: CD83 + Use of mesenchymal stem cells in the preparation of drugs for preventing / treating autoimmune diseases and / or inflammatory-related diseases.

[0011] Based on the heterogeneity and functional plasticity of MSCs, the team of this application believes that there are anti-inflammatory and pro-inflammatory subpopulations in MSCs, and the balance of pro / anti-inflammatory subpopulations is related to the outcome and treatment resistance of inflammatory / immune-related diseases. Therefore, if the cell surface markers and functional characteristics of the anti-inflammatory and pro-inflammatory subpopulations of MSCs can be clarified, and then high-purity MSCs anti-inflammatory subpopulations can be obtained through magnetic beads or flow cytometry sorting technology, or anti-inflammatory specialized MSCs can be removed from the pro-inflammatory subpopulation of MSCs, and then the anti-inflammatory subpopulation of MSCs or anti-inflammatory specialized MSCs are used as seed cells to develop new MSCs cell drugs specifically for the treatment of autoimmune / inflammatory-related diseases, it will greatly improve its clinical efficacy and have higher drugability. To this end, the team discovered a CD83-positive MSCs subpopulation through single-cell sequencing technology, and this subpopulation of cells has a more sensitive anti-inflammatory enabling effect on the immunosuppression or anti-inflammatory function mediated by inflammatory factors, and is defined as "immunosuppression or anti-inflammatory CD83 + -MSCs subpopulation", therefore, developing new MSCs cell drugs specifically for treating autoimmune / inflammatory related diseases based on this subpopulation of MSCs cells will greatly improve the clinical efficacy of MSCs and have higher drugability.

[0012] On the other hand, CD83 + Use of overexpressed mesenchymal stem cells in the preparation of drugs for preventing / treating autoimmune diseases and / or inflammatory-related diseases;

[0013] In certain specific embodiments, the CD83 + Mesenchymal stem cells / CD83 + Overexpression of mesenchymal stem cells can increase the secretion of anti-inflammatory factors and / or immunosuppressive factors.

[0014] In certain specific embodiments, the immunosuppressive factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3, and IL-2Rβ;

[0015] In certain specific embodiments, the anti-inflammatory factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ;

[0016] In certain specific embodiments, the autoimmune disease includes at least one of rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ankylosing spondylitis, myasthenia gravis and polycerebrospinal colitis; the inflammatory-related disease includes at least one of sepsis, degenerative arthritis and neonatal bronchopulmonary dysplasia pneumonia.

[0017] In the second aspect, a CD83 + Mesenchymal stem cells / CD83 + A method for preparing overexpression mesenchymal stem cells comprises the following steps:

[0018] (1) Isolate and culture mesenchymal stem cells;

[0019] (2) Using anti-CD83 antibody-coupled magnetic beads separation and flow cytometry, or lentiviral gene transduction technology, to isolate natural CD83 cells that are easily enabled to have immunosuppressive or anti-inflammatory functions. + Mesenchymal stem cells / CD83 + Overexpression of mesenchymal stem cells.

[0020] Specifically: Method 1 - magnetic beads and flow sorting technology: using anti-CD83 antibody coupled magnetic beads and flow sorting technology to sort out natural CD83 + Mesenchymal stem cells, wherein the sorting reagent includes magnetic beads coupled to anti-CD83 antibodies or CD83 flow cytometry sorting antibodies;

[0021] Or method 2 - lentiviral gene transduction technology: using lentiviral gene transduction technology to obtain artificial CD83 modified with human CD83 gene + Overexpression of mesenchymal stem cells, wherein the human CD83 gene transduction technology includes but is not limited to gene modification transduction technologies such as lentivirus, adenovirus and traditional plasmid expression vectors;

[0022] Preferably, the mesenchymal stem cells are derived from human adipose, dental pulp, bone marrow, umbilical cord, placenta or umbilical cord blood.

[0023] In certain specific embodiments, the step (1) of isolating and culturing mesenchymal stem cells comprises: collecting sample tissue and washing it; isolating the tissue; washing the separated tissue pieces in physiological saline, chopping them, weighing them, transferring them into a centrifuge tube, and centrifuging them, discarding the supernatant; adding MSCs culture medium to the centrifuge tube, and culturing them for a period of time; when the primary cells grow to a cell density of >80%, digesting and dissociating them, and subculturing them to obtain cells containing CD83 + Mixed mesenchymal stem cell populations of mesenchymal stem cells.

[0024] The third aspect: CD83 + PD-L1 +Use of mesenchymal stem cells in the preparation of drugs for preventing / treating autoimmune diseases and / or inflammatory-related diseases.

[0025] In certain specific embodiments, the CD83 + PD-L1 + Mesenchymal stem cells can enhance the secretion of anti-inflammatory factors and / or immunosuppressive factors.

[0026] In certain specific embodiments, the immunosuppressive factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3, and IL-2Rβ;

[0027] In certain specific embodiments, the anti-inflammatory factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ;

[0028] In certain specific embodiments, the autoimmune disease includes at least one of rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ankylosing spondylitis, myasthenia gravis and polycerebrospinal colitis; the inflammatory-related disease includes at least one of sepsis, degenerative arthritis and neonatal bronchopulmonary dysplasia pneumonia.

[0029] In the fourth aspect, a CD83 + PD-L1 + The preparation method of mesenchymal stem cells comprises the following steps: using an immune factor induction compound to induce and culture the aforementioned CD83 + Mesenchymal stem cells acquire CD83 with enhanced immunosuppressive or anti-inflammatory functions + PD-L1 + Mesenchymal stem cells.

[0030] In certain specific embodiments, the induction complex comprises IFN-γ and TNF-α immune factors, the concentration of the IFN-γ is 5-20 ng / ml, and the concentration of the TNF-α is 0-20 ng / ml.

[0031] Further, in certain specific embodiments, the induction complex comprises IFN-γ and TNF-α immune factors, the concentration of the IFN-γ is 20 ng / ml, and the concentration of the TNF-α is 5-20 ng / ml.

[0032] In certain specific embodiments, the induction complex further comprises an albumin excipient.

[0033] In certain specific embodiments, the timing of adding the induction complex is: 3-5 generations of CD83 + When the mesenchymal stem cells proliferate and culture to 70-80% cell confluence;

[0034] In certain specific embodiments, the induction culture specifically comprises: using immune factor induction complex to induce CD83 + The induction time of mesenchymal stem cells is 18 to 24 hours, and then the induction compound is withdrawn and the induced CD83 + Mesenchymal stem cells for at least 6 hours.

[0035] In the fifth aspect, a CD83 prepared according to the above preparation method + PD-L1 + Method for evaluating the quality of mesenchymal stem cells, the CD83 + PD-L1 + The positive expression rate of CD83 and PD-L1 receptors in mesenchymal stem cells was used as the CD83 + PD-L1 + Quality control indicators of mesenchymal stem cells.

[0036] In certain specific embodiments, the CD83 positive expression rate is at least ≥70%, and the PD-L1 receptor positive expression rate is ≥30%.

[0037] A kit, which can be effectively used to evaluate the positive expression rate of CD83 and PD-L1 receptors by using the above-mentioned quality assessment method + PD-L1 + Quality requirements of mesenchymal stem cells.

[0038] Compared with the prior art, the present invention has at least the following advantages:

[0039] 1) The present invention first discovered and verified CD83 + Mesenchymal stem cells are more sensitive to the expression and regulation of anti-inflammatory factors mediated by IFN-γ, which is manifested by the expression of higher levels of anti-inflammatory factors such as IL-10, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ after being stimulated by IFN-γ. + -MSCs subpopulation has a higher sensitivity to IFN-γ-mediated MSCs anti-inflammatory empowerment and is considered as an anti-inflammatory easily empowered subpopulation; in addition, CD83 +Mesenchymal stem cells acquire immunosuppressive or anti-inflammatory function-enhanced CD83 after induction by IFN-γ alone and / or in combination with TNF-α + PD-L1 + Mesenchymal stem cells, CD83 + PD-L1 + Mesenchymal stem cells can highly express immunosuppressive or anti-inflammatory factors such as PD-L1, IDO, IL-10, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ, and have extremely strong immunosuppressive or anti-inflammatory functions; Based on the present invention, the immunosuppressive or anti-inflammatory-enabling CD83 + -MSCs or CD83-enhanced immunosuppressive or anti-inflammatory functions induced by them + PD-L1 + -MSCs can treat inflammatory or immune-related diseases, because these two MSCs cells can highly express the above-mentioned anti-inflammatory factors and / or immunosuppressive factors, while eliminating the interference of pro-inflammatory MSCs subpopulations, thus playing a better immunosuppressive and anti-inflammatory effect on the treatment of immune and inflammatory-related diseases, leading to CD83 + Mesenchymal stem cells / CD83 + PD-L1 + Mesenchymal stem cells have better clinical efficacy in treating inflammatory or immune-related diseases. At the same time, the immunosuppressive or anti-inflammatory function-enhanced CD83 was preclinically evaluated using rheumatoid arthritis model mice and CLP model sepsis mice. + PD-L1 + -MSCs / CD83 cells that are easily enabled to have immunosuppressive or anti-inflammatory functions + Mesenchymal stem cells / CD83 + The effectiveness and safety of overexpressing mesenchymal stem cells in the treatment of immune or inflammatory diseases have been proven to be safe and effective.

[0040] 2) The present invention also found that the application of IFN-γ alone can upregulate CD83 at the transcriptional level. + -The expression of the immunosuppressive factor IDO and the immune checkpoint receptor PD-L1 in MSCs significantly enhanced the IFN-γ-mediated anti-inflammatory function of MSCs; and the combination of TNF-α and IFN-γ in the treatment of CD83 + -MSCs cells can significantly upregulate the protein expression of IDO and PD-L1 at both the transcriptional and translational levels, thereby enhancing the immunomodulatory and anti-inflammatory functions of MSCs. This invention innovatively proposes to use TNF-γ and / or IFN-α to induce the treatment of CD83 +-MSCs, and simultaneously upregulate the expression of the immunosuppressive factor IDO and the immune checkpoint receptor PD-L1 at the transcriptional and translational levels, thereby improving the immunomodulatory and anti-inflammatory capabilities of MSCs from different angles and enhancing their therapeutic efficacy in immune and inflammatory-related diseases.

[0041] 3) The present invention also successfully sorted or prepared natural or artificial CD83 that is easily capable of immunosuppressive or anti-inflammatory function by using anti-CD83 antibody-coupled magnetic bead separation and flow sorting technology, or lentiviral gene transduction technology. + Overexpression of mesenchymal stem cells. At the same time, on this basis, the aforementioned CD83 + Mesenchymal stem cells successfully obtained CD83 with enhanced immunosuppressive or anti-inflammatory function + PD-L1 + Mesenchymal stem cells.

[0042] 4) The present invention innovatively enhances the immunosuppressive or anti-inflammatory function of CD83 + PD-L1 + -MSCs can significantly reduce the mortality and inflammatory response of sepsis in the treatment of sepsis; it can also significantly improve the clinical symptoms of arthritis in mice by treating collagen-induced arthritis; at the same time, the present invention can also significantly improve the clinical symptoms of arthritis in mice by treating collagen-induced arthritis; + PD-L1 + -MSCs induction process, can further develop anti-inflammatory CD83 that can be easily promoted and applied + PD-L1 + -MSCs induction kit.

[0043] 5) In view of the role of PD-L1 signal in the anti-inflammatory function of MSCs and the fact that it is a membrane receptor that can be easily detected by flow cytometry, the detection of PD-L1 expression can be used as a quality control standard for the evaluation of the anti-inflammatory function of MSCs, which will provide a basis for the further development of anti-inflammatory CD83 + PD-L1 + -MSCs induction kit provided technical support. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0045] Figure 1 is the flow cytometry results of WT-MSCs surface marker molecules;

[0046] Figure 2 shows CD83 - 、CD83 + and WT-MSCs tri-lineage differentiation identification results;

[0047] Figure 3 shows the flow cytometry results of CD83 positive expression before and after MSCs sorting;

[0048] FIG4 is the detection result of human CD83 gene-modified CD83-overexpressing mesenchymal cells;

[0049] Figure 5 shows CD83 - and CD83 + -Flow cytometry detection results of MSCs cell surface markers;

[0050] Figure 6 shows CD83 - and CD83 + -MSCs cell proliferation ability test results;

[0051] Figure 7 shows CD83 - and CD83 + - MSCs cell clone proliferation ability test results;

[0052] Figure 8 shows CD83 - and CD83 + -MSCs cell migration ability test results;

[0053] Figure 9 shows CD83 - 、CD83 + and the results of in vitro anti-inflammatory function test and evaluation of WT-MSCs;

[0054] Figure 10 shows the results of gene and protein expression detection of IDO1 and PD-L1 in MSCs cells induced by different concentrations of TNF-α combined with IFN-γ;

[0055] Figure 11 shows TNF-α combined with IFN-γ to induce CD83 + -Time-dependent dynamic detection results of PD-L1 expression on MSCs membrane;

[0056] Figure 12 shows CD83 - 、CD83 + and WT-MSCs treatment efficacy evaluation results for rheumatoid arthritis;

[0057] Figure 13 shows natural CD83 + or overexpressing artificial CD83 + Results of the efficacy evaluation of overexpression of mesenchymal cells in the treatment of rheumatoid arthritis;

[0058] Figure 14 shows CD83 + or overexpressing artificial CD83 + Results of the efficacy evaluation of overexpression of mesenchymal cells in the treatment of sepsis;

[0059] Figure 15 shows the sham control group, PBS control group, WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE -Histopathological examination results of hematoxylin and eosin (H&E) staining of CLP-septic mice 72 h after MSCs treatment. DETAILED DESCRIPTION

[0060] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments and drawings. However, the following embodiments are only preferred embodiments of the present invention and are not exhaustive.

[0061] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0062] Example 1 Isolation, Culture and Identification of Human Umbilical Cord Mesenchymal Stem Cells

[0063] This example provides a process for establishing a preparation of human umbilical cord mesenchymal stem cells, specifically: isolating, culturing, and identifying human umbilical cord mesenchymal stem cells from the umbilical cord of a newborn, including the following steps:

[0064] (1) Isolation and culture of MSCs

[0065] Isolation and culture of human umbilical cord mesenchymal stem cells (hUC-MSCs): Fresh umbilical cord samples were collected under sterile conditions (samples were obtained from healthy pregnant women who tested negative for infectious diseases such as hepatitis B, hepatitis C, HIV, and syphilis, and had no genetic diseases, and had passed ethical application and obtained informed consent), and then washed with normal saline in an ultra-clean workbench in a GMP laboratory to clean blood residues and other substances on the surface of the sample. The sample was transferred to an alcohol dish and soaked for about 1 minute, and then transferred to a clean normal saline dish to wash off the alcohol on the umbilical cord; the two ends of the umbilical cord were cut off to remove the coagulated blood in the umbilical cord, and then the umbilical cord was cut into 2 cm umbilical cord blocks; the cut umbilical cord blocks were placed in a clean normal saline dish and washed again to further clean the coagulated blood in the umbilical cord. Liquid; Take a clean culture dish to separate Wharton's jelly; Use hemostatic forceps to remove the blood vessels (one vein and two arteries) in each section of umbilical cord in turn, and then separate the tissue skin from the tissue block along the edge with the skin side of the remaining umbilical cord tissue facing down (the tissue block is Wharton's jelly); Then place the separated colloid in a clean dish of physiological saline. After separating the colloid, use tweezers to break up the large Wharton's jelly as much as possible. Finally, wash the colloid again in a clean dish of physiological saline and transfer the Wharton's jelly to a sterile vial that has been disinfected; Use scissors to cut the Wharton's jelly into as small pieces as possible and weigh it. Divide it into 15ml centrifuge tubes by weight, 0.5g per tube, add 10ml of physiological saline, centrifuge at 2000rpm for 5 minutes, and discard the supernatant; Take two 25CM 2 Culture flask, marked with the primary culture number, culture date and number or barcode; add 4ml serum-free Ultra CULTURE to each of the two centrifuge tubes TM Culture medium (containing 2mM L-glutamine) (add 4ml of culture medium for every 0.5g of tissue block); cover the lid and shake well, place the culture flask in an incubator (37°C, 5% CO2 incubator), and culture for 5 to 7 days, then add fresh culture medium; when the primary cells grow to a cell density ≥80%, digest the adherent cells with TrypLE, centrifuge and replace the medium before subculturing; the hUC-MSCs used in the present invention are 3rd to 5th generation cells as the original seed bank cells.

[0066] (2) Identification of mesenchymal stem cells

[0067] ① Identification of surface marker molecules of mesenchymal stem cells:

[0068] Flow cytometry was used to detect the expression of surface markers in the isolated and cultured mesenchymal stem cells. Flow cytometry analysis detected positive stem cell markers CD73, CD44, CD90, and CD105, as well as negative markers CD29, CD34, and CD45. The results showed that the hUC-MSCs purified and cultured in vitro were positive for CD44, CD73, CD90, and CD105, and negative for CD29, CD34, and CD45, meeting the International Society for Cellular Therapy's MSC definition. The results of flow cytometry analysis for negative and positive expression of surface markers are shown in Figure 1.

[0069] ② Identification of the three-lineage differentiation ability of mesenchymal stem cells:

[0070] Osteogenic Differentiation: Wild-type hUC-MSCs at passage 3 were digested and counted, then seeded into six-well plates. A defined volume of complete culture medium was added to each well. When the cell density reached 60%-70%, the medium was replaced with OriCell Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation Medium. Fresh culture medium was replaced periodically based on cell growth. After approximately 2-4 weeks of culture, 1 mL of Alizarin Red stain was added to each well. The plates were washed twice with PBS and then observed under a microscope for osteogenic staining.

[0071] Chondrogenic differentiation: wild-type third generation hUC-MSCs were taken and 4×10 5 Transfer the cells to a centrifuge tube, centrifuge and discard the supernatant; add 0.5mL of the kit premix (the kit premix can be purchased through commercial channels. The kit premix in this embodiment is one of the components of the human umbilical cord mesenchymal stem cell chondrogenic differentiation kit purchased from OriCell, model number HUXUC-90041), resuspend the hUC-MSCs pellet, centrifuge again at room temperature, and discard the supernatant; then resuspend the cells with 0.5mL of OriCell human umbilical cord mesenchymal stem cell chondrogenic differentiation complete medium, seed the cells into six-well plates, and then place them in an incubator for culture; replace the cells with fresh chondrogenic differentiation complete medium every once in a while. After continuous induction culture for 28 days, the chondrocytes were formalin-fixed and paraffin-embedded in sections, stained with alicein blue dye, washed with PBS to remove excess dye, and observed under a microscope to evaluate the chondrogenic staining effect.

[0072] Adipogenic differentiation: Wild-type hUC-MSCs at passage 3 were digested and counted, then seeded into six-well plates. A certain amount of complete medium was added to each well and the cells were cultured in an incubator until the cell density reached 100%. The medium in each well was replaced with OriCell Human Umbilical Cord Mesenchymal Stem Cell Adipogenic Differentiation Medium A. After 72 hours of induction, the medium A in the six-well plate was aspirated and OriCell Human Umbilical Cord Mesenchymal Stem Cell Adipogenic Differentiation Medium B was added. After 24 hours, the medium B was aspirated and replaced with medium A for induction. After alternating culture with medium A and medium B for 3-5 cycles (approximately 12-20 days), the cells were maintained in medium B (the medium was changed every 3 days) for 7 days. The cells were then fixed with paraformaldehyde for 30 minutes, washed with PBS, and stained with 1 mL of Oil Red O working solution per well. The excess dye was washed off with PBS, and the adipogenic staining effect was evaluated under a microscope.

[0073] The results of the identification of the tri-lineage differentiation ability of wild-type mesenchymal stem cells (WT-MSCs) are shown in Figure 2. As can be seen from Figure 2, the wild-type hUC-MSCs obtained in the present invention (labeled as WT-MSCs in the figure) have the ability to differentiate into osteocytes, chondrocytes and adipocytes under specific induction culture conditions, indicating that the wild-type hUC-MSCs obtained by culture and purification in the present invention meet the standards of the International Society for Cell Therapy for human MSCs.

[0074] Example 2 CD83 + -Sorting, preparation, culture and identification of MSCs

[0075] This example establishes CD83 + Preparation process of anti-inflammatory and easily empowered human umbilical cord mesenchymal stem cell subsets.

[0076] From the wild-type human umbilical cord mesenchymal stem cell seed bank, CD83 was obtained by magnetic bead or flow cytometry sorting or gene modification overexpression. + The specific steps for inhibiting inflammation and empowering human umbilical cord mesenchymal stem cells are as follows:

[0077] (1)CD83 + Sorting and preparation of mesenchymal stem cells:

[0078] ① Magnetic bead separation of CD83 +Mesenchymal stem cells: Based on Example 1, LS sorting columns were used in combination with a magnetic sorter; hUC-MSCs were cultured to a confluency of 80%, and the hUC-MSCs cell suspension was obtained by 0.25% trypsin digestion. The cells were washed with PBS 1-2 times, and the PBS was discarded by centrifugation. The hUC-MSCs cells were incubated with anti-CD83 monoclonal antibodies in the dark for 15 minutes, and the non-specifically bound anti-CD83 antibodies were washed away. Magnetic beads were then added and incubated in the dark for another 10 minutes. The incubated sample was passed through the column. The cells labeled with magnetic beads were adsorbed on the sorting column under the action of the magnetic field, and the negative cells that were not bound to the anti-CD83 antibodies flowed into the test tube, which was CD83. - -MSCs; remove the LS separation column from the magnetic stand, rinse the separation strain again, and the hUC-MSCs cells bound to the magnetic beads and anti-CD83 antibodies are eluted due to the lack of magnetic force, thereby obtaining CD83 positive MSCs, i.e. CD83 + -MSCs; cells were collected by centrifugation, and a portion was identified and separated by flow cytometry to identify CD83 + -The positive expression ratio of CD83 on the cell membrane surface of MSCs, as the CD83 + -MSCs cell sorting purity index, the remaining cells were cultured for another generation, and then the purity was identified by flow cytometry analysis. If the purity was ≥80%, the cells were frozen and stored as CD83 + Figures 3A-3B show the CD83 positive expression ratio on the membrane surface of hUC-MSCs before (Figure 3A) and after (Figure 3B) magnetic bead sorting. The ratio before sorting was about 15%, and the purity after sorting exceeded 98%, indicating that magnetic bead sorting can obtain high-purity CD83 + -MSCs.

[0079] ② or flow cytometry sorting of CD83 + Mesenchymal stem cells: In addition to using magnetic bead separation to obtain high-purity CD83 + -MSCs can also be obtained by flow cytometry or other sorting techniques. + -MSCs, the sorting scheme is as follows: FITC or PE fluorescently labeled anti-CD83 antibody is incubated with hUC-MSCs at room temperature for 30 minutes, and the cells are resuspended into a single cell suspension. Then, the flow cytometry sorting parameters are set according to the operating requirements of the flow cytometer, and the cells are loaded and sorted to obtain high-purity CD83 + -MSCs cells were cultured for one generation and the purity was determined by flow cytometry. If the purity was ≥80%, the cells were frozen and stored as CD83 + -MSCs cell seed bank.

[0080] ③ Gene modification of human CD83 to generate artificial CD83-overexpressing mesenchymal stem cells: LV-CD83 lentivirus was used to construct stable CD83-overexpressing mesenchymal cells. The viral vector sequence consisted of the following: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin. The CD83 gene sequence was retrieved from GenBank (Gene ID: NM_004233.4). The LV-CD83 lentiviral expression vector was constructed, packaged, and purified for use. Before transfection, MSCs were suspended in complete medium and seeded into T25 culture flasks. The cells were cultured at 37°C, 5% CO₂ for 16-24 hours until the cell confluency reached approximately 40% before lentiviral transfection. Either HiTransG A or HiTransG P infection medium was selected. The appropriate amount of virus was added based on the cell MOI and viral titer (MOI: 10 in this experiment). The cells were then cultured in a 37°C, 5% CO₂ incubator for 16 hours before being replaced with complete medium and continued culturing. Approximately 72 hours after infection, the efficiency of viral infection was observed under a fluorescence microscope. Because lentiviral-infected cells are resistant to puromycin, 1 mg / ml puromycin was added to kill uninfected cells. CD83-overexpressing cell lines were purified and screened. After screening, cells were passaged and cryopreserved for future use. Figure 4A shows the fluorescence microscopy observation of CD83 overexpression with GFP fluorescence, and Figure 4B shows the results of immunoblotting using an anti-Flag tag antibody to detect CD83 protein expression in MSCs.

[0081] (2)CD83 + Identification of Mesenchymal Stem Cells:

[0082] ①CD83 + Identification of surface marker molecules of mesenchymal stem cells: The implementation steps are as shown in (2)-① in Example 1, and the mesenchymal stem cells are replaced with CD83 + or CD83 - Mesenchymal stem cells can be prepared according to the above steps. - or CD83 + There was no difference in surface marker expression between hUC-MSCs and wild-type hUC-MSCs: they were positive for CD44, CD73, CD90, and CD105, and negative for CD29, CD34, and CD45, meeting the MSC criteria defined by the International Society for Cellular Therapy. The flow cytometry results for negative / positive expression of surface markers are shown in Figure 5.

[0083] ②CD83 + Identification of the three-lineage differentiation ability of mesenchymal stem cells: The implementation steps are as shown in (2)-② in Example 1, and wild-type mesenchymal stem cells are replaced with CD83 - or CD83+ -MSCs can be implemented according to the above steps. The results of the three-lineage differentiation experiment are shown in Figure 2. - or CD83 + -MSCs and wild-type mesenchymal stem cells hUC-MSCs (labeled as WT-MSCs in the figure) are consistent in that they have the ability to differentiate into osteocytes, chondrocytes and adipocytes under specific induction culture conditions, indicating that the CD83 + -MSCs, which also meet the MSCs standards defined by the International Society for Cellular Therapy.

[0084] ③CD83 + Identification of mesenchymal stem cell proliferation function: Based on the principle that Ki-67 protein is a cell cycle-related nuclear protein that is mainly expressed during the cell proliferation and division phase, but not expressed in resting cells (G0 phase), the detection of Ki-67 content can reflect the cell proliferation activity. In addition, phosphorylated histone H3 (PH3) is highly expressed during cell mitosis and can be used as a stable indicator of cell proliferation. CD83 - -MSCs and CD83 + -MSCs cells were digested with 0.25% trypsin and prepared into single cell suspension. Sterile cleaned coverslips were added to 6-well plates and then 1×10 5 The amount of cells / well was CD83 - -MSCs and CD83 +-MSCs cells were seeded in 6-well plates and cultured for 24 hours. When the cell confluence reached 60-70%, the coverslips with cells were removed, washed twice with PBS, and fixed with 4% paraformaldehyde for 1h-2h; a few drops of PBS were added to the fixed cell slides to cover them for 15 minutes, and then washed three times with PBS; cell permeabilization solution (0.1% Triton-X) was added to the slides and placed on ice for 5 minutes; the slides were soaked in detergent for 5 minutes, washed three times, and then hydrogen peroxide working solution was added to cover the samples and placed on ice for 5-10 minutes; the slides were soaked in PBS for 2 minutes, washed three times, and then 50μl of ready-to-use goat serum blocking solution was added to each slide. After incubation in a 37℃ wet box for 10 minutes, the slides were soaked in detergent for 2 minutes and washed three times; a slide with anti-Ki67 and PH3 100 μL of anti-Ki-67 and PH3 antibodies (both diluted at 1:100) were added and incubated in a wet box at 37°C for 1 hour; the samples were soaked in PBS for 2 minutes, washed 3 times, and then 50 μL of ready-to-use HRP-labeled secondary antibody IgG was added to cover the samples and incubated in a wet box at 37°C for 10 minutes; the samples were soaked in PBS for 2 minutes, washed 5 times, and then one drop of prepared DAB color development solution was added to each slide and color was developed at room temperature for 2-5 minutes; the samples were soaked in PBS for 2 minutes, washed 5 times, and then the staining depth was observed under a microscope. The staining was stopped immediately, and the slides were gently rinsed with tap water for 15 minutes and the color development reaction was terminated with distilled water. The slides were then stained for DNA with DAPI at a final concentration of 100 ng / ml for 3 minutes, soaked in PBS for 2 minutes, washed 5 times, sealed, and examined under an optical microscope and photographed. Figure 6A shows the results of Ki67 staining of cell slides, Figure 6B shows the results of PH3 staining of cell slides, and Figure 6C shows the Ki67 and PH3 positive statistical results of Figures 6A and 6B. As shown in the results of Figures 6A-6C, under the condition of no IFN-γ stimulation, CD83 - -MSCs and CD83 + -MSCs had weak proliferation ability between the two groups and no significant difference, but after stimulation by IFN-γ, the proliferation ability of the two groups was weaker than that of CD83 - Compared with CD83-MSCs, + -The proliferation ability of MSCs was significantly enhanced.

[0085] ④CD83 + Identification of mesenchymal stem cell clone formation ability: CD83 - -MSCs and CD83 + -MSCs cells were digested with 0.25% trypsin and prepared into single cell suspension. About 200 CD83 - -MSCs and CD83 +-MSCs cells, blow the cells apart or rotate them manually to make them evenly dispersed, and then place them in a cell culture incubator for static culture for 2-3 weeks. When small balls that can be distinguished by the naked eye appear in the culture dish, discard the supernatant, wash with PBS, fix for 10 minutes, stain with crystal violet for 30 minutes, wash with PBS, and take pictures for observation. As shown in Figures 7A and 7B, the clonal proliferation capacity of MSCs with and without IFN-γ stimulation is shown in Figures 7A and 7B. - -MSCs and CD83 + There was no significant difference in the clonal proliferation ability between the two groups of MSCs, but after stimulation by IFN-γ, the CD83 - Compared with CD83-MSCs, + -The clonal proliferation ability of MSCs was significantly enhanced.

[0086] ⑤CD83 + Identification of mesenchymal stem cell migration function:

[0087] (a) Cell scratch assay: CD83 in the logarithmic growth phase was digested with 0.25% trypsin. - -MSCs and CD83 + -MSCs cells, made into 5×10 5 A single-cell suspension of 100 cells / ml was plated in a 6-well plate, with 2 ml of the cell suspension in each well. The cells were cultured in a cell culture incubator for 24 hours. When the confluence reached 100%, a straight line was drawn using a 100 μl pipette tip (using a ruler for assistance). Care was taken to ensure uniform and straight lines were drawn. The scratched cells were washed with PBS, and fresh culture medium was replaced with IFN-γ. The cells were then cultured in a cell culture incubator. Images were taken at 0, 6, 12, and 24 hours after the scratch, and the scratch area was calculated using Image J software.

[0088] (b) Transwell assay: CD83 in the logarithmic growth phase was digested with 0.25% trypsin. - -MSCs and CD83 + -MSCs cells, prepared 1×10 5 A single-cell suspension of 10 cells / ml was prepared and placed in a Transwell well with 500 μl of suspension in each well. The Transwell was placed in a 24-well plate and 1 ml of culture medium and stimulatory factor IFN-γ were added to each well of the 24-well plate. The cells were cultured in a cell culture incubator for 24 hours, the culture medium was discarded, the cells were fixed with fixative for 10 minutes, washed twice with PBS, stained with crystal violet for 30 minutes, washed twice with PBS, and the cell migration was observed under a microscope.

[0089] Figure 8A shows the microscope photography results of the cell scratch assay at 0 hours and 24 hours, Figure 8B shows the statistical results of the scratch area in Figure 8A, Figure 8C shows the crystal violet staining results of the lower layer cells after 24 hours of Transwell assay, and Figure 8D shows the statistical results of the crystal violet staining positive cells in Figure 8C. As shown in the results of Figures 8A-8D, both the scratch assay and the Transwell assay showed that in the absence of IFN-γ, although CD83 + -MSCs are more sensitive to CD83 - -MSCs showed better migration ability, but there was no statistical difference between the two groups. However, after IFN-γ stimulation, CD83 + -MSCs are more sensitive to CD83 - -The migration ability of MSCs was significantly enhanced.

[0090] Example 3: CD83 with enhanced immunosuppression or anti-inflammatory function + PD-L1 + Induction, culture and identification of double-positive mesenchymal stem cells

[0091] The CD83 prepared in Example 2 was induced by IFN-γ alone and / or TNF-α in combination. + -MSCs, acquire CD83 with enhanced immunosuppressive or anti-inflammatory function + PD-L1 + -MSCs, the specific steps are:

[0092] 1. In vitro induction of immunosuppressive or anti-inflammatory function-enhanced CD83 + PD-L1 + MSCs

[0093] The WT-MSCs, CD83 + -MSCs and CD83 - -MSCs cryopreserved cells were revived and cultured at 4×10 4 cells / cm 2 The cells were inoculated into T75 culture flasks at a density of 100 μg / mL and placed in an incubator (37°C, 5% CO2). When the cells grew to a cell density of ≥80%, they were digested with 0.25% TrypLE and the digested cells were plated at 4×10 4 cells / cm 2The cells were seeded at a density of 100 μg / ml in T25 culture flasks for passage and expansion, and the cells were divided into single and combined treatment groups in the factor inducers of IFN-γ (0, 5, 10, 20 ng / ml) and / or TNF-α (0, 5, 10, 20 ng / ml). The culture flasks were placed in a 37°C, 5% CO2 incubator and cultured until the cell density was >80%. The above-mentioned factor inducers (IFN-γ (0, 5, 10, 20 ng / ml) and / or TNF-α (0, 5, 10, 20 ng / ml)) were added for induction for 24 hours, and then the cells were harvested by digestion with 0.25% TrypLE. The cell sample protein and mRNA were extracted, and the expression of the anti-inflammatory factor spectrum was detected by inflammatory protein chip. The expression of the cellular immunosuppressive factor IDO and the receptor PD-L1 was detected by RT-PCR, immunoblotting and flow cytometry. The mixed lymphocyte experiment was used to evaluate the inhibitory function of MSCs cells on the proliferation of T lymphocytes, so as to comprehensively evaluate the CD83 + -MSCs have immunosuppressive or anti-inflammatory functions.

[0094] 2. Inflammatory protein chip detection of immunosuppressive or anti-inflammatory function enhanced CD83 + PD-L1 + Expression profile of anti-inflammatory factors in MSCs

[0095] Extract CD83 treated with the above different stimulating factors + -MSCs and CD83 - -MSCs protein samples; the prepared protein samples were tested on inflammatory protein chips and compared with CD83 + -MSCs and CD83 - -The expression of anti-inflammatory factors in MSCs was different after treatment with 20ng / ml IFN-γ alone.

[0096] As shown in Figure 9A, CD83 + -MSCs and CD83 - -MSCs cell subsets showed significant differences in the expression of inflammatory / anti-inflammatory factors after IFN-γ treatment. + -MSCs are more sensitive to the expression of anti-inflammatory factors mediated by IFN-γ, as shown by the significant increase in the expression of anti-inflammatory factors such as IL-10, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ, suggesting that CD83 + -MSCs subpopulation is more sensitive to IFN-γ-mediated MSCs anti-inflammatory empowerment and can be regarded as an anti-inflammatory easily empowered subpopulation.

[0097] 3. Quantitative PCR detection of immunosuppressive or anti-inflammatory function-enhanced CD83+ PD-L1 + mRNA expression of immunosuppressive factor IDO and immunosuppressive receptor PD-L1 in MSCs

[0098] Quantitative PCR detection technology was used to detect the expression of immunosuppressive factor IDO and immunosuppressive receptor PD-L1 mRNA in mesenchymal stem cells prone to immunosuppression or anti-inflammatory function.

[0099] Figures 9B-9C show the quantitative PCR results of 20ng / ml IFN-γ alone inducing MSCs to express IDO1 and PD-L1. The quantitative PCR results of Figures 9B-9C show that WT-MSCs, CD83 - -MSCs, CD83 + When the three groups of MSCs were not stimulated by IFN-γ, the basal expression of the immunosuppressive factor IDO and the immunosuppressive receptor PD-L1 mRNA remained at extremely low levels, with no significant difference between them. However, when MSCs were stimulated by IFN-γ alone for 24 hours, the mRNA expression of IDO1 and PD-L1 in the three groups of cells was significantly upregulated, and compared with CD83 - -MSCs, CD83 + The expression of IDO1 and PD-L1 mRNA in -MSCs increased most significantly, followed by WT-MSCs.

[0100] Figures 10A-10B show the quantitative PCR test results of MSCs expressing IDO1 and PD-L1 induced by different concentrations of TNF-α combined with IFN-γ. As shown in the figure, 5, 10 and 20 ng / ml of TNF-α alone could not induce the transcription of IDO1 and PD-L1, and mRNA was only slightly increased. 5, 10 and 20 ng / ml of IFN-γ alone could significantly induce the increase of IDO1 and PD-L1 mRNA, especially 20 ng / ml of IFN-γ could induce PD-L1 mRNA to increase by 85 times. However, interestingly, unlike the results of immunoblotting, TNF-α combined with IFN-γ stimulation did not further increase the mRNA expression of IDO1 and PD-L1. On the contrary, 20 ng / ml of TNF-α slightly weakened the mRNA expression of IDO1 and PD-L1 induced by IFN-γ.

[0101] 4. Immunoblotting to detect CD83 with enhanced immunosuppressive or anti-inflammatory functions + PD-L1 + IDO and PD-L1 protein expression in MSCs

[0102] Extract WT-MSCs, CD83 +-MSCs and CD83 - -MSCs protein sample; performing polyacrylamide gel electrophoresis (SDS-PAGE) on the prepared protein sample; and then performing Western blot analysis on the gel after electrophoresis.

[0103] Figures 9D-9F show the immunoblotting results of 20ng / ml IFN-γ alone inducing the expression of IDO1 and PD-L1 in MSCs, where Figures 9E and 9F are the immunoblotting densitometric analysis results of IDO1 and PD-L1 (also known as CD274) in Figure 9D. The immunoblotting results in Figures 9D-9F show that IFN-γ alone stimulates MSCs for 24 hours, which can induce the protein expression of IDO1 and PD-L1 (CD274), and the expression of CD83 is significantly higher than that of MSCs. - -MSCs, CD83 + -MSCs expressed more significant IDO1 and PD-L1.

[0104] Figure 10C shows the results of immunoblotting of IDO1 and PD-L1 expression in MSCs induced by different concentrations of TNF-α combined with IFN-γ. The immunoblotting results in Figure 10C show that 20 ng / ml TNF-α alone stimulated MSCs for 24 hours and induced neither IDO1 nor PD-L1 expression. However, when MSCs were treated with 20 ng / ml IFN-γ combined with different concentrations of TNF-α, the results showed that the addition of 5 ng / ml TNF-α induced the expression of both IDO1 and PD-L1, and the expression of IDO1 and PD-L1 gradually increased with increasing TNF-α concentration.

[0105] 5. Flow cytometry detection of immunosuppressive or anti-inflammatory function-enhanced CD83 + PD-L1 + Expression of PD-L1, a cellular immune checkpoint receptor, in MSCs

[0106] CD83 + MSCs were cultured to 80% confluency and then treated with 20 ng / ml IFN-γ alone or in combination with 20 ng / ml, 40 ng / ml, 60 ng / ml, 80 ng / ml, and 100 ng / ml TNF-α and negative control PBS for 24 h. After that, they were digested with 0.25% TrypLE, washed with physiological saline, and resuspended to prepare single-cell suspension (1 × 10 6 PD-L1 FITC was added to the cell suspension and incubated at room temperature in the dark for 30 minutes; washed twice with PBS before flow cytometry analysis.

[0107] FIG10D shows that IFN-γ alone or in combination with TNF-α induces CD83 + -Flow cytometry results of PD-L1 expression in MSCs. As shown in Figure 10D, CD83 + -MSCs were induced with 20ng / ml IFN-γ alone for 24 hours, and the flow cytometric fluorescence intensity (MFI) of PD-L1 expression increased by about 55%. When combined with 10ng / ml TNF-α for 24 hours, the flow cytometric fluorescence intensity (MFI) of PD-L1 expression increased significantly by about 145%. As the TNF-α treatment concentration increased to 20ng / ml and 40ng / ml, the flow cytometric fluorescence intensity (MFI) of PD-L1 expression increased slightly. However, when the TNF-α treatment concentration exceeded 60ng / ml, the increased fluorescence intensity of PD-L1 expression was inhibited. This suggests that IFN-γ combined with TNF-α treatment can enhance the expression of the immunosuppressive receptor PD-L1, and the optimal combined concentration of TNF-α is between 10ng / ml and 40ng / ml.

[0108] Figure 11A ①-④ represent the induction of CD83 by no induction factor, 20ng / ml TNF-α alone, 20ng / ml IFN-γ alone, and 20ng / ml TNF-α and 20ng / ml IFN-γ combined. + -MSCs cell PD-L1 expression flow cytometry results after 24 hours; Figure 11B ①-④ respectively represent no induction factor, 20ng / ml TNF-α induction alone, 20ng / ml IFN-γ induction alone, and 20ng / ml TNF-α and 20ng / ml IFN-γ combined induction of CD83 + After 24 hours of culture, the stimulating factors were withdrawn and the MSCs were cultured for another 24 hours. The results of flow cytometry analysis of PD-L1 expression were obtained. In Figure 11C, ①-④ represent the expression of CD83 in the absence of induction factors, induction with 20 ng / ml TNF-α alone, induction with 20 ng / ml IFN-γ alone, and induction with 20 ng / ml TNF-α and 20 ng / ml IFN-γ, respectively. + After 24 hours of MSCs culture, the stimulating factors were withdrawn and the MSCs were cultured for another 48 hours. The results of PD-L1 expression were detected by flow cytometry. As shown in Figures 11A-11C, 20 ng / ml TNF-α alone induced CD83 + -MSCs, whether stimulated for 24 hours or cultured for 24 or 48 hours after stimulation, the expression of the immunosuppressive receptor PD-L1 was not affected. +-MSCs, the expression of the immunosuppressive receptor PD-L1 was slightly increased after 24 hours of stimulation, but after 24 hours of stimulation and then cultured for 24 or 48 hours, the expression of PD-L1 increased significantly, reaching 31.94% at 48 hours. + -MSCs can significantly and rapidly increase the expression of PD-L1. 24 hours after stimulation, the positive rate of PD-L1 expression can quickly reach 25.85%. If cultured for another 24 hours and 48 hours, the positive rates reach 33.55% and 61.23% respectively, almost reaching 1 times that induced by IFN-γ alone.

[0109] 6. Flow cytometry detection of immunosuppressive or anti-inflammatory function-enhanced CD83 + PD-L1 + Inhibitory effect of MSCs on PBMC proliferation

[0110] In 1×10 7 1 μl (1000×) of CFSE fluorescent dye was added to each peripheral blood mononuclear cell (PBMCs) for nuclear staining. After incubation in the dark for 20 minutes, the cells were washed twice with PBS. PBMCs not stained with CFSE were used as negative controls, and PBMCs not co-cultured were stained as positive controls. The stained PBMCs were then incubated with CD83 T cells treated with 20 ng / ml IFN-γ for 24 hours. - -MSCs, CD83 + -MSCs and WT-MSCs were co-cultured, and IL-2 was added to each well of the co-culture system at a final concentration of 100 IU / ml. After 3 days of co-culture, the changes in the CFSE fluorescence values ​​of PBMCs in each group were detected by flow cytometry to determine the proliferation inhibition effect of PBMCs. Figure 9G shows the changes in intracellular CFSE fluorescence values ​​after co-culture of PBMCs and MSCs by flow cytometry, and Figure 9H shows the PBMC cell proliferation inhibition rate calculated from Figure 9G. As shown in Figures 9G and 9H, the CD83 - -MSCs, CD83 + -MSCs and WT-MSCs can significantly inhibit the proliferation of PBMCs. + -MSCs had the strongest inhibitory effect on the proliferation of PBMCs, followed by WT-MSCs, among which CD83 - -MSCs had the worst inhibitory effect.

[0111] Example 4 Immunosuppressive or anti-inflammatory function is easily endowed to CD83 +Application and efficacy evaluation of human umbilical cord mesenchymal stem cells in the treatment of collagen-induced arthritis in mice

[0112] 1. Establishment of collagen-induced arthritis (CIA) model in mice and MSCs treatment plan and process

[0113] A CIA mouse model was constructed according to the literature (the construction of CIA mouse model is a prior art and therefore will not be described in detail here). At the same time, in order to reduce individual differences in animals and better evaluate the therapeutic effect, when the arthritis score reached or exceeded 3 points after the booster injection, MSCs treatment was started after the onset of the disease. At the same time, WT-MSCs and CD83 + -MSCs and CD83 - -MSCs seed cells were revived and cultured for 48 hours until the cell confluence reached 80%, and then the cells were harvested and MSCs cell preparations were prepared. As shown in Figure 12A, a single tail vein infusion of 1×10 6 Wild-type human umbilical cord mesenchymal stem cells (WT-MSCs group, treatment control group), CD83 + -MSCs, CD83 - -MSCs and PBS group (non-treatment control group). The intervention time of the collagen model mice selected in this application was MSCs tail vein injection after the onset of arthritis (approximately within 3-5 days after the booster injection). On the 50th day, the mice were tested for the pain threshold of thermal pain and mechanical pain. In addition, on the 50th day, the animals were sacrificed, and blood and limb joints were collected for flow cytometry analysis, cytokine detection and histopathological examination, and imaging examination evaluation was performed at the same time.

[0114] Clinical Scoring Protocol for Arthritis in the CIA Mouse Model: CIA mice were evaluated for arthritis using a clinical scoring system based on paw swelling. Clinical arthritis was assessed using the following scale: 0, no swelling; 1, mild swelling and erythema; 2, marked edema; and 3, joint stiffness. Each limb was scored and summed, with a maximum possible score of 12 per animal.

[0115] 2. CD83 is prone to immunosuppression or anti-inflammatory function + -Evaluation of the therapeutic effect of MSCs in a collagen-induced arthritis mouse model

[0116] ①Gross observation and swelling score of arthritis in CIA mice treated with MSCs

[0117] According to the clinical scoring standard of arthritis of plantar swelling, the swelling degree of each limb of mice in each treatment group was scored. Figures 12B (gross observation) and 12D (swelling degree score) show the differences between the negative control group (NC) and the mice injected intravenously with PBS, WT-MSCs, and CD83+ -MSCs and CD83 - -MSCs-induced CIA mice arthritis swelling assessment results. The CIA model was successfully established after collagen induction for about 25 days. MSCs were injected on the 27th day and the joint swelling was scored. The results showed that WT-MSCs, CD83 + -MSCs and CD83 - -MSCs three kinds of MSCs can significantly improve the swelling of the toes of CIA mice, and in comparison, CD83 + -MSCs had the most significant anti-edema effect on CIA arthritis mice and the lowest index score.

[0118] ②Imaging evaluation of MSCs in treating arthritis in CIA mice

[0119] At the end of the experiment (day 50), the hind limbs of CIA mice were imaged using vivaCT 40 small animal CT (SCANO MEDICAL, Switzerland) and Bruker BioSpec 7T / 20 cm system small animal Magnetic Resonance Imaging (MRI) (Bruker, Germany).

[0120] Figure 12C shows the negative control group (NC) and mice injected intravenously with PBS, WT-MSCs, CD83 + -MSCs and CD83 - -MSCs-treated CIA mice. Small animal MRI and small animal CT were used to image the hind limbs of CIA mice on day 50, and representative images are shown. At least 5 mice were used in each group: NC = negative control (n = 5 mice), PBS = CIA mice PBS-treated group (n = 10 mice), WT-MSCs = CIA mice WT-MSCs-treated group (n = 10 mice), CD83 + -MSCs = CIA mouse CD83 + -MSCs treatment group (n=10 mice), CD83 - -MSCs = CIA mouse CD83 - -MSCs treatment group (n = 10 mice), all results are mean ± SD. As shown in Figure 12B (CT detection results) and 12C (MRI detection results), compared with the PBS treatment control, a single intravenous injection of WT-MSCs, CD83 + -MSCs and CD83 - -MSCs three kinds of MSCs can significantly improve synovitis, arthritis, cartilage damage and bone destruction in CIA mice, and in comparison, CD83+ -MSCs group had the best therapeutic effect, followed by WT-MSCs, CD83 - -MSCs treatment had the worst efficacy.

[0121] ③Evaluation of pain threshold in arthritic toes of CIA mice treated with MSCs

[0122] At the end of the experiment (day 50), the thermal nociceptive response was assessed using a hot plate analgesia instrument to obtain the thermal pain threshold of CIA mice; the paw withdrawal threshold of the hind paw surface of mice was measured using the Von Frey fiber pressure method to obtain the mechanical pain threshold of CIA mice.

[0123] Figures 12E and 12F show the negative control group (NC) and mice injected intravenously with PBS, WT-MSCs, and CD83 + -MSCs and CD83 - Results of thermal pain threshold and mechanical pain threshold examination of CIA mice treated with WT-MSCs. As shown in Figure 12E (thermal pain threshold) and 12F (mechanical pain threshold), compared with the PBS-treated control, a single intravenous injection of WT-MSCs, CD83 + -MSCs and CD83 - -MSCs three kinds of MSCs can significantly improve the thermal pain threshold and mechanical pain threshold of CIA mice, and in comparison, CD83 + -MSCs group had the best improvement in pain threshold, CD83 - -MSCs treatment showed the worst improvement.

[0124] ④Evaluation of MSCs in treating systemic and toe inflammation in CIA mice with arthritis

[0125] At the end of the experiment (day 50), the mice were killed, and the serum and local soft tissue of the toes were collected. The levels of TNF-α and IL-6 in the serum and soft tissue were detected by ELISA to determine the systemic and local anti-inflammatory effects of MSCs on CIA mice.

[0126] Figures 12G and 12H show the results of ELISA detection of inflammatory factors TNF-α and IL-6 in the serum of CIA mice treated with three cell lines on day 50; Figures 12I and 12J show the results of ELISA detection of inflammatory factors TNF-α and IL-6 in the local tissues of the limbs and soles of CIA mice treated with three cell lines on day 50. As shown in Figures 12G-12J, compared with the negative control, the inflammatory mediators TNF-α and IL-6 in the serum and soft tissues of CIA mice were significantly increased. Compared with the PBS treatment group, the WT-MSCs, CD83 + -MSCs and CD83 -After single treatment with three types of MSCs, the levels of inflammatory mediators TNF-α and IL-6 in serum and soft tissue of CIA mice were significantly reduced. + -MSCs group had the most significant anti-inflammatory effect, CD83 - -MSCs group had the worst anti-inflammatory effect.

[0127] ⑤ Histopathological evaluation of MSCs-treated arthritis in CIA mice

[0128] On the 50th day of the experiment, all mice were killed, and the limb joint tissues of the mice were collected. The limb specimens fixed with 4% paraformaldehyde were decalcified with a decalcification solution containing 15% EDTA, and then the decalcified limbs were dehydrated and paraffin-embedded according to standard histological methods.

[0129] a) Hematoxylin-eosin (H&E) staining: After routine xylene dewaxing and gradient alcohol hydration, the sections were immersed in warm hematoxylin stain for 2 minutes, the stain was quickly rinsed with water, hydrated with 1% hydrochloric acid alcohol for 15 seconds, and then rinsed again with water. The staining effect was observed under a microscope. The standard was that the nucleus was blue-purple and the intercellular matrix was not stained. Then eosin staining was performed for 1 minute, followed by gradient alcohol dehydration, xylene transparentization, and neutral gum sealing. H&E staining was used to assess the severity of arthritis: 0 points for normal synovium, 1 point for synovial hypertrophy and cell infiltration, 2 points for vascular membrane and cartilage erosion, 3 points for cartilage and subchondral bone erosion, and 4 points for loss of joint integrity and ankylosis. The evaluation was performed by multiple third-party testers, and the average of the scores of multiple testers was used as the final value.

[0130] b) Safranin O and Fast Green staining: Deparaffinize and rehydrate with water, then treat with 0.02% Fast Green for 5 minutes (not rinsed), 1% acetic acid for 30 seconds (not rinsed), and 0.1% Safranin O for 20 minutes (not rinsed). Rinse in 95% ethanol for 2 minutes, followed by two rinses in 100% alcohol for 3 minutes each. Then, rinse in xylene for 2 minutes each. Cover with a coverslip. Sections were then stained with Safranin O and Fast Green to assess cartilage destruction.

[0131] Figure 12K shows the results of histopathological evaluation of mice sacrificed on day 50. Paraffin-embedded sections of the metacarpal and posterior phalangeal joints of CIA mice were stained with H&E, Safranin O, and Fast Green, respectively, to average their histological joint damage and cartilage destruction, and representative images were selected. Scale bar = 200 μm. At least 5 mice were used in each group: In Figure 12K, NC is the negative control (n = 5 mice), PBS is the CIA mouse PBS-treated group (n = 10 mice), WT-MSCs is the CIA mouse WT-MSCs-treated group (n = 10 mice), CD83 +-MSCs for CIA mice CD83 + -MSCs treatment group (n=10 mice), CD83 - -MSCs for CIA mice CD83 - -MSCs treatment group (n=10 mice). As shown in Figure 12K, compared with the PBS treatment group, WT-MSCs, CD83 + -MSCs and CD83 - -MSCs three kinds of MSCs can significantly reduce the experimental arthritis joint synovitis, joint destruction and cartilage destruction, compared with CD83 + -MSCs group showed the most significant improvement in osteoarthritis, CD83 - -MSCs group had the worst improvement effect. + -MSCs and CD83 - -MSCs-treated mice did not experience any side effects or die before the termination of the experiment.

[0132] Example 5 Application and efficacy evaluation of artificial CD83-overexpressing mesenchymal stem cells endowed with immunosuppressive or anti-inflammatory functions in the treatment of collagen-induced arthritis mice

[0133] 1. Establishment of collagen-induced arthritis (CIA) model in mice and MSCs treatment plan and process

[0134] The CIA mouse model was established according to Example 4, and the artificial CD83 overexpressing mesenchymal stem cells were prepared according to Example 2, that is, the wild-type human umbilical cord mesenchymal stem cells (WT-MSCs) obtained in Example 1 and the CD83 overexpressing mesenchymal stem cells (CD83-MSCs) screened in Example 2 were used. + Human umbilical cord mesenchymal stem cells (CD83 + -MSCs) were used as seed cells, and lentiviral overexpression technology was used to obtain CD83 overexpressing WT cells. CD83 OE -MSCs and CD83 + / OE -MSCs. The therapeutic effect of artificial CD83 overexpressing mesenchymal stem cells on CIA mice arthritis was evaluated according to the MSCs treatment plan and process in Example 4. The animals were divided into negative control group normal mice (NC) and intravenously injected PBS, CD83 + / GFP -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs in CIA mice.

[0135] 2. Evaluation of the efficacy of immunosuppressive or anti-inflammatory function-endowed artificial CD83-overexpressing mesenchymal stem cells in the treatment of arthritis in CIA mice

[0136] The efficacy evaluation includes gross observation and swelling score of arthritis, pain threshold evaluation, systemic and toe local inflammation evaluation, imaging and pathological evaluation. For specific evaluation methods and measures, please refer to Example 4. Figure 13 shows the evaluation of the therapeutic effect of artificial CD83 overexpressing mesenchymal stem cells on arthritis in CIA mice. Figure 13A shows the process of artificial CD83 overexpressing mesenchymal stem cells treating arthritis in CIA mice; Figures 13B and 13D show the gross redness and swelling observation and swelling score of the toe joints of CIA mice treated with artificial CD83 overexpressing mesenchymal stem cells. The results show that compared with the PBS treatment group, CD83 + / GFP -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs cells in the three groups all had therapeutic effects on arthritis in mice, and the joint redness and swelling and scores were significantly improved. + / OE -MSCs and WT CD83 OE -MSCs cells in the two groups had the most significant improvement on joint redness and swelling and scores, and there was no significant difference between the two groups; Figure 13C shows the CT and MRI evaluation results of the toe joints of CIA mice treated with artificial CD83 overexpressing mesenchymal stem cells. The results showed that compared with the PBS treatment group, CD83 + / GFP -MSCs, CD83 + / OE -MSCs and WT CD83 OE The three groups of MSCs cells showed significant improvement effects on synovitis, arthritis, cartilage damage and bone destruction in CIA mice, among which CD83 overexpressed + / OE -MSCs and WT CD83 OE -MSCs cells had the most significant therapeutic effects on arthritis, and there was no significant difference between the two groups; Figures 13E and 13F respectively evaluated the thermal pain threshold and mechanical pain threshold of CIA mice treated with artificial CD83-overexpressing mesenchymal stem cells. The results showed that CD83 + / GFP -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs in all three groups could increase the pain threshold of CIA mice, and CD83 overexpression + / OE -MSCs and WT CD83 OE-MSCs cells showed the most significant improvement in pain threshold in CIA mice, and there was no significant difference between the two groups; Figures 13G and 13H show the ELISA test results of inflammatory factors TNF-α and IL-6 in the serum of CIA mice treated with artificial CD83-overexpressing mesenchymal stem cells on day 50; Figures 12I and 12J show the ELISA test results of inflammatory factors TNF-α and IL-6 in the local tissues of the limbs and soles of CIA mice treated with artificial CD83-overexpressing mesenchymal stem cells on day 50. As shown in Figures 13G-13J, compared with the negative control, the inflammatory mediators TNF-α and IL-6 in the serum and soft tissues of CIA mice were significantly increased. Compared with the PBS-treated group, the CD83-overexpressing mesenchymal stem cells showed a significant increase in the levels of TNF-α and IL-6 in the serum and soft tissues of CIA mice. + / GFP -MSCs, CD83 + / OE -MSCs and WT CD83 OE After a single treatment with MSCs, the levels of inflammatory mediators TNF-α and IL-6 in serum and soft tissue of CIA mice were significantly reduced. + / OE -MSCs and WT CD83 OE -MSCs had the most significant anti-inflammatory effect in the two groups, and there was no significant difference between the two groups.

[0137] Example 6: CD83 is susceptible to immunosuppression or anti-inflammatory function + Application and efficacy evaluation of human umbilical cord mesenchymal stem cells in the treatment of CLP sepsis in mice

[0138] 1. Establishment of CLP model in septic mice and MSCs treatment plan and process

[0139] BALB / c mice aged 8 weeks and weighing about 22-25 g were selected for cecal ligation and puncture (CLP). Three hours after the operation, 0.2 mL of sterile PBS was injected into the tail vein of the sham control group and the PBS treatment control group. WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs group were injected with 2×10 5 The cells were suspended in 0.2 mL of cell suspension, and imipenem and cilastatin sodium (14 mg / Kg) were added after 24 hours. The mortality rate of each group was finally calculated at 168 hours. The serum samples for liver function, renal function, cardiac function and systemic inflammatory index examination and the tissue samples for pathological examination were collected 72 hours after surgery. The sample collection process was as follows: BALB / c mice underwent cecal ligation and puncture (CLP) 3 hours later, the sham operation control group and the PBS treatment control group were injected with 0.2 mL of sterile PBS through the tail vein, and WT-MSCs, CD83 + -MSCs, CD83 + / OE-MSCs and WT CD83 OE -MSCs group were injected with 2×10 5 200 μl of cell suspension containing 100 cells was prepared. 24 hours later, imipenem and cilastatin sodium (14 mg / kg) were added with antibiotics. 72 hours after CLP, mice in each group were anesthetized with 1% sodium pentobarbital (40 mg / kg) intraperitoneally, and their eyes were enucleated for blood collection. Vital organs and tissues were then dissected and collected. Whole blood was allowed to rest at room temperature for 2 hours. After blood coagulation and stratification, the blood was centrifuged at 1500 rpm for 10 minutes, three times. Serum was collected and assayed for levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 using ELISA. Liver function (including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), renal function (including serum creatinine, blood urea nitrogen (Urea), and amylase (AMS), and cardiac function (including lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (α-HBDH), and creatine kinase (CK)) were assessed in CLP mice using conventional biochemical assays. The tissue specimens were processed according to the pathological tissue examination protocol in Example 4, and then subjected to H&E staining for histopathological examination.

[0140] The preparation process of the MSCs cells used in this Example 6 was carried out according to the methods and steps of Example 5, except that 48 hours before harvesting, each group of MSCs was treated with 20 ng / ml IFN-γ and 20 ng / ml TNF-α according to the scheme in Example 3 for PD-L1 positive expression anti-inflammatory stimulation for 24 hours, and then switched to a medium without IFN-γ and TNF-α and continued to culture for 24 hours. The cells were harvested for subsequent septic mouse treatment experiments.

[0141] 2. CD83 is prone to immunosuppression or anti-inflammatory function + Protective effect of human umbilical cord mesenchymal stem cells on the survival of CLP-induced sepsis mice

[0142] Figure 14 shows that WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs treatment of CLP sepsis mice. As shown in Figure 14A, the sham control group (sham), PBS control group, WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE-MSCs group treated CLP sepsis mice survival rate 168 hours after treatment, the results showed that the four groups of anti-inflammatory MSCs cells can significantly improve the survival rate of CLP sepsis mice, in comparison, CD83 overexpression of CD83 + / OE -MSCs and WT CD83 OE -MSCs MSCs in both groups had the best protective effect on mouse survival, and there was no difference between the two groups. + The protective effect of the -MSCs group on the survival of CLP sepsis mice was significantly better than that of the WT-MSCs group.

[0143] 3. CD83 is prone to immunosuppression or anti-inflammatory function + Protective effects of human umbilical cord mesenchymal stem cells on organ function in CLP-induced sepsis mice

[0144] Figures 14B-14I show sham control group, PBS control group, WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs treatment improved liver function (including aspartate aminotransferase AST and alanine aminotransferase ALT), renal function (including serum creatinine Creatinine, blood urea nitrogen Urea and amylase AMS) and cardiac function (including lactate dehydrogenase LDH, α-hydroxybutyrate dehydrogenase α-HBDH and creatine kinase CK) in CLP sepsis mice 72 hours later. The results showed that compared with the negative control, the liver, kidney and heart functions of CLP sepsis mice (PBS control group) were severely damaged, and the above indicators were significantly increased. After treatment with the above four anti-inflammatory MSCs, the above indicators reflecting liver, kidney and heart functions were restored to varying degrees. In comparison, CD83 overexpressed CD83 + / OE -MSCs and WT CD83 OE -MSCs MSCs had the best effect on improving the liver, kidney and heart function of mice, and there was no difference between the two groups. + -MSCs group had a significantly better effect on improving liver, kidney and heart functions in CLP septic mice than WT-MSCs group.

[0145] 4. CD83 is prone to immunosuppression or anti-inflammatory function + Anti-inflammatory effect of human umbilical cord mesenchymal stem cells on CLP-induced sepsis in mice

[0146] Figures 14J-14L show sham control group, PBS control group, WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE-MSCs treatment had an anti-inflammatory effect on CLP sepsis mice 72 hours later. The results showed that compared with the negative control, the inflammatory indicators IL-1β, IL-6 and TNF-α in the serum of CLP sepsis mice (PBS control group) were significantly increased. After treatment with the above four anti-inflammatory MSCs, the levels of IL-1β, IL-6 and TNF-α in the serum were significantly decreased. In contrast, CD83 overexpressed + / OE -MSCs and WT CD83 OE -MSCs group and MSCs group had the best inhibitory effect on the above three inflammatory factors, and there was no difference between the two groups. In terms of the inhibition of inflammatory factor expression, CD83 + -MSCs group was slightly better than WT-MSCs group.

[0147] 5. CD83 is prone to immunosuppression or anti-inflammatory function + Protective effects of human umbilical cord mesenchymal stem cells on liver and lung tissue damage in CLP-induced sepsis mice

[0148] Figures 15A-15B show sham control group, PBS control group, WT-MSCs, CD83 + -MSCs, CD83 + / OE -MSCs and WT CD83 OE -MSCs treatment of CLP sepsis mice 72 hours after the hematoxylin and eosin (H&E) staining of the histopathological examination results showed that compared with the negative control, CLP sepsis mice (PBS control group) had damaged liver lobule structure, liver parenchyma showed a large number of inflammatory cell infiltration; alveolar wall thickening, a large number of inflammatory cells infiltrated into the lung interstitium and alveolar space, and a large amount of exudate appeared in the alveoli. After treatment with the above four anti-inflammatory MSCs, liver and lung structural damage was alleviated, inflammatory cell infiltration in the tissues was reduced, and CD83 overexpression was significantly reduced. + / OE -MSCs and WT CD83 OE -MSCs had the best protective effect on liver and lung tissue damage in the two groups, and there was no difference between the two groups.

[0149] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. 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 make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. CD83 + Use of mesenchymal stem cells in the preparation of a medicament for preventing / treating autoimmune diseases and / or inflammation-related diseases.

2. CD83 + Use of overexpressing mesenchymal stem cells in the preparation of a medicament for preventing / treating autoimmune diseases and / or inflammation-related diseases.

3. CD83 according to claim 1 or 2 + Mesenchymal stem cells / CD83 + Application of overexpressed mesenchymal stem cells It is characterized in that The CD83 + Mesenchymal stem cell / CD83 + Overexpressing mesenchymal stem cells can increase the secretion of anti-inflammatory factors and / or immunosuppressive factors.

4. The application according to claim 3, It is characterized in that The anti-inflammatory factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ.

5. The application according to claim 3, It is characterized in that The immunosuppressive factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ.

6. The application according to claim 1 or 2, It is characterized in that The autoimmune diseases include at least one of rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ankylosing spondylitis, myasthenia gravis and multiple ulcerative colitis, etc.; the inflammation-related diseases include at least one of sepsis, degenerative arthritis and neonatal bronchopulmonary dysplasia pneumonia, etc.

7. CD83 in the application according to any one of claims 1-6 + Mesenchymal stem cells / CD83 + Method for preparing mesenchymal stem cells with overexpression It is characterized in that It includes the following steps: (1) Isolate and culture mesenchymal stem cells; (2) Use magnetic beads conjugated with anti-CD83 antibody and flow sorting technology, or lentiviral gene transduction technology to sort and obtain immunosuppressive or anti-inflammatory function easily empowered natural CD83 + Mesenchymal stem cell / CD83 + Overexpressed mesenchymal stem cells.

8. The CD83 according to claim 7 + Method for preparing mesenchymal stem cells It is characterized in that The mesenchymal stem cells are derived from human adipose tissue, dental pulp, bone marrow, umbilical cord, placenta or umbilical cord blood. 9.CD83 + PD-L1 + Use of mesenchymal stem cells in the preparation of a medicament for preventing / treating autoimmune diseases and / or inflammation-related diseases.

10. The application according to claim 9, It is characterized in that The CD83 + PD-L1 + Mesenchymal stem cells can enhance the secretion of anti-inflammatory factors and / or immunosuppressive factors.

11. The application according to claim 10, It is characterized in that The anti-inflammatory factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ.

12. The application according to claim 10, It is characterized in that The immunosuppressive factor is at least one of IL-10, IDO, PD-L1, GDNF, IL-10Rα, IL-2Rγ, IL-4, IL-4R, IL-17B, LIF, GM-CSFRα, TIMP-3 and IL-2Rβ.

13. The application according to claim 9, It is characterized in that The autoimmune diseases include at least one of rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ankylosing spondylitis, myasthenia gravis and multiple ulcerative colitis, etc.; the inflammation-related diseases include at least one of sepsis, degenerative arthritis and neonatal bronchopulmonary dysplasia pneumonia, etc.

14. A CD83 + PD-L1 + Preparation method of mesenchymal stem cells It is characterized in that Including inducing and culturing the CD83 described in claim 1 with an immune factor induction complex + mesenchymal stem cells to obtain CD83 with enhanced immunosuppressive or anti-inflammatory function + PD-L1 + mesenchymal stem cells.

15. The CD83 according to claim 14 + PD-L1 + Method for preparing mesenchymal stem cells It is characterized in that The induction complex includes IFN-Υ and TNF-α immune factors, the concentration of IFN-Υ is 5-20 ng / ml, and the concentration of TNF-α is 0-20 ng / ml.

16. The CD83 according to claim 15 + PD-L1 + Method for preparing mesenchymal stem cells It is characterized in that The induction complex includes IFN-Υ and TNF-α immune factors, the concentration of IFN-Υ is 20 ng / ml, and the concentration of TNF-α is 5-20 ng / ml.

17. The CD83 according to claim 15 + PD-L1 + Method for preparing mesenchymal stem cells It is characterized in that The induction complex further includes an albumin excipient.

18. A CD83 prepared by the preparation method according to any one of claims 14-17 + PD-L1 + A method for quality assessment of mesenchymal stem cells It is characterized in that Taking the positive expression rates of CD83 and the PD-L1 receptor in the mesenchymal stem cells as the quality control index of the CD83 + PD-L1 + Taking the positive expression rates of CD83 and the PD-L1 receptor in the mesenchymal stem cells as the quality control index of the CD83 + PD-L1 + Taking the positive expression rates of CD83 and the PD-L1 receptor in the mesenchymal stem cells as the quality control index of the mesenchymal stem cells.

19. The quality assessment method according to claim 19, characterized in that: the positive expression rate of CD83 is at least ≥70%, and the positive expression rate of the PD-L1 receptor is ≥30%.

20. A kit, characterized in that, The kit can effectively be used to evaluate the quality requirements of CD83 + PD-L1 + mesenchymal stem cells by detecting the positive expression rates of CD83 and PD-L1 receptors using the quality assessment method described in claim 18 or 19.

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