Preparation of CD146 + MSC cells and application of CD146 + MSC cells in treatment of type 2 diabetes mellitus

Abstract

The invention belongs to the technical field of medicines, and particularly relates to preparation of CD146 + MSC cells and application of the CD146 + MSC cells in treatment of type 2 diabetes mellitus. Researches find that the CD146 + MSC has better pluripotency in the aspect of being differentiated into fat cells, bone cells and cartilage cells; the proliferation speed is low; the in-vivo and in-vitro immunosuppressive activity is enhanced. Meanwhile, the CD146 + MSC has higher differentiation capacity and immunosuppressive activity, the CD146 + MSC has higher dryness, lower telomerase activity and higher migration and homing capacities, shows higher biological characteristics of immunosuppression and cytokine secretion, and is remarkable in proliferation and angiogenesis activity, so that the CD146 + MSC has better immunosuppressive activity and immunosuppressive activity. A cell type with higher stemness and more original characteristics is represented. In addition, the CD146 + MSC cells can treat type 2 diabetes mellitus by reconstructing beta cells, enhancing and regulating blood glucose homeostasis, relieving insulin resistance, and reducing or regulating systemic inflammation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to CD146 + Preparation of MSC cells and its application in treating type 2 diabetes. BACKGROUND

[0002] Type 2 diabetes (T2D) is characterized by insulin resistance (IR) in insulin-sensitive tissues and impaired insulin secretion by pancreatic beta cells. With disease progression, beta cell function decreases by 6-8% per year, which plays a key role in the development and progression of type 2 diabetes. Studies have shown that the pathogenesis of type 2 diabetes (T2DM) is related to systemic low-grade chronic inflammation. First, excessive nutrition leads to the proliferation of adipose tissue, which causes an imbalance between pro-inflammatory and anti-inflammatory cytokines, and thus promotes the infiltration of immune cells. Subsequently, inflammatory factors and chemotactic factors in adipose tissue are released into the blood circulation, affecting various tissues including beta cells, adipose tissue, liver and muscle, ultimately leading to deterioration of insulin sensitivity and beta cell function.

[0003] Traditional T2D treatment usually includes lifestyle modification and oral hypoglycemic drugs. However, many patients gradually find it difficult to achieve ideal glycemic control, resulting in the need for these patients to eventually use insulin to lower blood glucose levels, and T2D patients usually need to take these drugs for life. Therefore, attention must be paid to the possible adverse reactions of the drugs. More importantly, these treatments can only temporarily lower blood glucose levels, but have no long-term effect on diabetes and its complications such as irreversible pancreatic beta cell damage. Therefore, it is extremely urgent to explore new methods to cure diabetes and maintain therapeutic effect.

[0004] Mesenchymal stem cells (MSCs) have been extensively and intensively explored in preclinical and clinical studies. These cells, with their ability to modulate the microenvironment (such as immunomodulation and tissue regeneration function), show great potential in the treatment of various diseases. However, due to the heterogeneity of MSCs, although the number of clinical trials of MSCs has increased rapidly in recent years, only a few have achieved the expected clinical effect.

[0005] The heterogeneity of mesenchymal stem cells (MSCs) and the inconsistency of therapeutic effects seriously hinder their application in clinical practice. Among them, the source, processing method, health status and genetic background of the donor can all lead to heterogeneity. Studies have shown that purifying specific MSC subpopulations to achieve specific therapeutic purposes is another promising strategy to reduce MSC heterogeneity and improve the consistency of therapeutic effects. Some markers of human MSCs have been identified, including Stro-1, CD166, CD271, CXCR4, GD2, CD146, CD200, CD49f, PODXL, CD140a, Lgr6, Lgr5, ROR2, CD264, CD143 and CD362. However, these markers are not specific to MSCs. In addition, the specific mechanisms of these markers on their functions are not fully understood. Therefore, it is necessary to further identify more MSC-specific markers.

[0006] CD146, also known as melanoma cell adhesion molecule (MCAM) or cell surface glycoprotein (MUC18), is a cell adhesion molecule involved in angiogenesis. CD146 can be widely expressed in MSCs from various sources, such as bone marrow, adipose tissue, umbilical cord, synovial membrane, and dermis. Sorrentino et al. (Hu X, Garcia M, Weng L, et al. Identification of a common mesenchymal stromal progenitor for the adult hematopoietic niche [J]. Nature communications, 2016, 7(1): 13095.) found that CD146 + MSCs represent a subset of stromal cells that support hematopoiesis and secrete complex growth factors to control the function of hematopoietic stem cells (HSCs), so CD146 + MSCs can be used to explore the hematopoietic stem cell "niche" in MSC-HSC in vitro replacement models and regenerative therapy research. However, although a variety of MSC subpopulations have been discovered, few MSC-specific markers have been identified. Among the functions of MSCs, immune regulation is the most concerned function, especially in the biology and therapeutic applications of MSCs. Analysis shows that CD146 is a potential MSC marker. CD146 + The functions of MSCs are related to immune regulation. Therefore, it is necessary to further explore CD146 + The multipotency and immunosuppressive ability of MSCs can expand their application field. SUMMARY

[0007] In order to overcome the above deficiencies of the prior art, the present application provides a DMEM culture medium containing 5% human platelet cell lysate, which can be used for expanding human CD146 + MSCs while maintaining their characteristics and functions. On this basis, it is disclosed that CD146 + MSCs have cell population diversity and MSC heterogeneity caused by batch differences of cell expansion culture medium; and in an acute inflammatory mouse model, CD146 + MSC subpopulation is identified to have enhanced immunosuppressive activity and therapeutic effect. In addition, CD146 + MSCs can play a therapeutic role on type 2 diabetes by rebuilding beta cells, enhancing and regulating blood glucose homeostasis, relieving insulin resistance, and reducing or regulating systemic inflammation.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is: The present application provides CD146 + MSC cells in the preparation of a medicament for treating type 2 diabetes.

[0009] The present application discloses that CD146 + MSC cells can treat type 2 diabetes (T2D) by rebuilding beta cells, enhancing and regulating blood glucose homeostasis, relieving insulin resistance, and reducing or regulating systemic inflammation, thereby providing a new approach for the control of T2D.

[0010] Preferably, CD146 + The preparation method of the MSC cells is as follows: first, human mesenchymal stem cells (MSCs) are extracted from umbilical cords and expanded using a DMEM culture medium containing 5% human platelet cell lysate, then stained with anti-CD146-PE or IgG-PE antibodies after subculture, and finally purified using a cell sorter.

[0011] More preferably, the MSCs are subcultured to the 3rd generation and used for CD146 + MSC cell purification.

[0012] More preferably, the MSCs are dissociated using TrypLE before antibody staining.

[0013] More preferably, after the human umbilical cord is cut into pieces, it is digested with 1-2 mg / mL collagenase B, and then MSCs are obtained after expansion in a DMEM culture medium containing 5% human platelet cell lysate.

[0014] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0015] More preferably, the adjuvants include at least one of excipients, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, antioxidants, adsorbents, filtration aids, release retardants.

[0016] More preferably, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations or suppositories.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application finds that CD146 + The MSC cells have stronger stemness and more primitive characteristics, specifically, the pluripotency in adipocyte, osteocyte and chondrocyte differentiation is more optimal, the migration and homing ability is stronger, the proliferation and angiogenesis activity is significantly higher, the telomerase activity is lower, the in vivo and in vitro immunosuppression activity is enhanced, and the cytokine secretion biological characteristics are more optimal; more importantly, the cells can effectively treat type 2 diabetes by rebuilding beta cells, enhancing and regulating blood glucose homeostasis, relieving insulin resistance and reducing or regulating systemic inflammation, thereby providing a more optimal cell selection and potential solution for the treatment of related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 CD146 + MSC cells and CD146 - Characteristics of MSC cells: A: cell morphology under light microscope, showing CD146 + MSC and CD146 - MSC under ordinary optical microscope; B: immunofluorescence staining diagram, showing the expression of CD146 in cells by fluorescence staining (blue is generally cell nucleus staining), to determine the presence or absence of CD146 in the two kinds of cells; C: quantitative analysis diagram related to fat differentiation, including Oil Red O staining OD 500quantitative analysis of the mRNA levels of the key genes of adipogenic differentiation (LPL, PPARy) at different times (Day 7, 21) to compare the adipogenic differentiation abilities of the two kinds of cells; D: Oil Red O staining photomicrograph of adipogenic differentiation, which directly presents the formation of lipid droplets (red staining) after adipogenic differentiation induction of the two kinds of cells, and reflects the differences in adipogenic differentiation efficiency; E: quantitative analysis chart related to osteogenic differentiation, which covers Alizarin Red staining OD569 value quantification (detecting calcium nodule formation), quantitative analysis of the mRNA levels of the key genes of osteogenic differentiation (OSTERIX, RUNX2) at different times (Day 7, 21) to compare the osteogenic differentiation abilities of the two kinds of cells; F: Alizarin Red staining photomicrograph of osteogenic differentiation, which directly presents the formation of calcium nodules (red staining) after osteogenic differentiation induction of the two kinds of cells, and reflects the differences in osteogenic differentiation efficiency; G: quantitative analysis (relative mRNA levels of SOX9 and BMP2) and quantitative results chart (OD 600 value) of Alizarin Blue staining related to chondrogenic differentiation; H: Alizarin Blue staining chart (50 pm) of chondrogenic differentiation; I: Alizarin Blue staining chart (200 pm) of chondrogenic differentiation; J: statistical column chart of chondrogenic efficiency; K: statistical column chart related to cell proliferation, which shows CD146 + MSC and CD146 - Cell counting of MSC, which reflects the differences in proliferation speed.

[0019] Figure 2 CD146 + MSC cells and CD146 - Results of immunosuppression research of MSC cells: A: column chart for presenting the relative OD - values of MSC, CD146 + MSC in MSC-PBMC co-culture experiments, which reflects the situation related to lymphocyte proliferation inhibition, and compares the levels of lymphocyte proliferation inhibition of different mesenchymal stem cells and the differences in IFN-gamma simulation experiments; B: HE staining chart of tissue sections, which shows the morphological structure of LPS-induced mouse acute inflammation model after PBS, CD146 600 MSC, CD146 - MSC treatment; C: column chart for counting the number of CD45 + lymphocytes in the lung tissues of LPS-induced mouse acute inflammation model of different treatment groups (PBS, CD146 - MSC, CD146 + MSC), which reflects the effect of CD45 + lymphocyte inhibition; D: column chart for counting the number of CD3 +The effect of lymphocyte infiltration; D: bar chart, statistical analysis of the effects of different treatment groups (PBS, CD146) in an LPS-induced acute inflammation model in mice. - MSC, CD146 + MSC (neutrophil count) reflects the effect of neutrophil infiltration; E: bar chart, detecting the different treatment groups (PBS, CD146) in an LPS-induced mouse acute inflammation model. - MSC, CD146 + MSC myeloperoxidase activity was used to reflect neutrophil-related immune status; F: bar chart, detecting the activity of different treatment groups (PBS, CD146) in an LPS-induced acute inflammation model in mice. - MSC, CD146 + The levels of pro-inflammatory cytokines such as IL-6, TNF-α, IFN-γ, and IL-1β in the serum of MSCs were measured to demonstrate immunosuppressive effects at the cytokine level.

[0020] Figure 3 CD146 + Figure A: Experimental results related to the in vivo and in vitro distribution, targeting, and migration of MSCs; A: Whole-body fluorescence imaging of mice. After tail vein injection of DIR-labeled cells, NC (blank control), MSCs, and CD146 were observed at different time points of 2h, 6h, and 12h. + Fluorescence distribution of MSCs in mice; B: Fluorescence imaging of the pancreas, with separate imaging of the pancreas after dissection, compared with NC, MSC, and CD146. + Fluorescence in the pancreas of the MSC group; C: Quantitative histogram of pancreatic fluorescence intensity. Quantitative statistical analysis of pancreatic fluorescence in Figure B was performed to identify CD146. + The pancreatic fluorescence in the MSC group was significantly higher than that in other groups, demonstrating its targeting specificity to the pancreas; D: Blood fluorescence imaging at different time points, detecting NC, MSC, and CD146 at 1h, 3h, and 6h. + MSC group blood fluorescence was used to observe cell retention in the bloodstream; E: quantitative histogram of blood fluorescence intensity, D: quantitative blood fluorescence, and data presentation of CD146. + The MSC group showed higher blood fluorescence, indicating a longer blood circulation time; F: Fluorescence imaging of organs such as the heart, liver, lungs, spleen, and kidneys, multi-organ imaging after dissection, compared with CD146. + Fluorescence in various organs of MSCs and MSC groups helps to observe differences in cell distribution within the body; G: Fluorescent staining image of cell migration, using MIN-6 cells (pancreatic β-cell model) as the subject, fluorescent labeling (DIO) to observe migration to MSCs and CD146. +Migration of MSC, green color for migrated MIN-6 cells, blue color for nucleus staining (DAPI); H: flow cytometry quantification of cell migration, quantification of migrated MIN-6 cells by flow cytometry, different colors represent NC, MSC, CD146 + MSC group; I: quantification of cell migration, further statistics of flow cytometry results of H, and CD146 is presented by column chart + MSC group has significantly higher migration amount, which proves that MSC promotes the migration of pancreatic beta cells.

[0021] Figure 4 CD146 + Results of the experiment of the protective effect of MSC on the islets of T2D mice; A: schematic diagram of the experimental process, showing the modeling of T2D mice and the timeline of receiving different treatments (PBS, MSC, CD146 + MSC); B-D, F: line graphs, respectively presenting the changes of random blood glucose (B), body weight (C), fasting and glucose tolerance test (IPGTT) blood glucose (D), and insulin sensitivity related blood glucose (F) over time or detection nodes, for intuitive comparison of the blood glucose data differences of different treatment groups (Control+PBS, T2D+PBS, T2D+MSC, T2D+CD146 + MSC); E, G, H, I, L, M: column chart (E, G, L), box plot (I), scatter box plot (M), and multi-group data comparison column chart (H) for presenting the quantitative statistical results at specific time points or indicators, wherein E is the quantification of IPGTT 30min blood glucose, G is the quantification of insulin sensitivity, H is the comparison of pancreatic islet area and other indicators, I is the box plot statistics of pancreatic islet area, L is the column statistics of insulin related cell proportion, and M is the scatter box plot statistics of pancreatic beta cell area; J: HE staining diagram of pancreatic tissue, showing the morphological structure of pancreatic tissue by staining, and comparing the morphological differences of islets in different treatment groups; K: immunofluorescence staining diagram of pancreatic tissue, using fluorescent markers such as insulin and glucagon to show the distribution and expression of related cells in the islets. DETAILED DESCRIPTION

[0022] The specific embodiments of the present application will be further described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0024] Although preclinical and clinical studies have shown that mesenchymal stem cells (MSCs) have great application potential in the treatment of various diseases, the problem of inconsistent therapeutic effects caused by cell heterogeneity has become a major obstacle to their clinical application. In order to reduce the heterogeneity of mesenchymal stem cells and improve the therapeutic effect and consistency, the relevant research field has carried out a lot of exploration. Among the many coping strategies, purifying homogenous mesenchymal stem cell populations by enhancing biological functions is considered as a promising method.

[0025] A variety of human MSC subpopulations have been found, but few MSC-specific markers. Immune regulation is the most concerned function of MSCs, especially in their biological research and therapeutic applications. Analysis shows that CD146 is a potential MSC marker, and CD146 + MSCs are associated with immune regulation. Therefore, the present application aims to further explore CD146 + The multipotency characteristics, immunosuppressive ability and potential mechanisms of MSCs.

[0026] The research data of the present application show that CD146 + MSCs have better multipotency in differentiating into adipocytes, osteocytes and chondrocytes; slower proliferation rate; enhanced immunosuppressive activity in vivo and in vitro. At the same time, CD146 + MSCs have stronger differentiation ability and immunosuppressive activity, CD146 + MSCs have stronger stemness, lower telomerase activity, stronger migration and homing ability, exhibit stronger immunosuppressive and cytokine secretion biological characteristics, and have significant proliferation and angiogenic activity, representing a cell type with higher stemness and more primitive characteristics. In addition, CD146 + MSCs can treat type 2 diabetes by reconstituting beta cells, enhancing and regulating glucose homeostasis, relieving insulin resistance, and reducing or regulating systemic inflammation.

[0027] In order to comprehensively and clearly present the technical solutions of the present application and its significant advantages, the present application will be described in detail below in combination with specific embodiments.

[0028] 1. Experimental methods 1.1. Isolation, expansion and characterization of human mesenchymal stem cells (MSCs) This study was approved by the Ethics Committee of the Second People's Hospital of Shenzhen and followed the principles of the Declaration of Helsinki. Human umbilical cords were minced and digested with 1 mg / mL collagenase B and cultured in DMEM medium containing 5% human platelet lysate (PLT Gold Clinical Grade, Mill Creek Life Sciences) for purification expansion, after which MSCs were collected. Differentiation and characterization of MSCs were performed using differentiation kits, including StemPro® Adipogenic Differentiation Kit (Gibco), StemPro® Osteogenic Differentiation Kit (Gibco), and StemPro® Chondrogenic Differentiation Kit (Gibco), respectively.

[0029] 1.2, Flow cytometry Cell preparation, antibody staining, and analysis for flow cytometry were performed according to the BD Biosciences protocol. Briefly, mesenchymal stem cells (MSCs) were treated with TrypLE, resuspended in PBS containing 5% BSA, and incubated with antibodies against CD146-PE, CD73-FITC, CD90-FITC, CD105-FITC, CD45-FITC, CD34-FITC, CD19-FITC, CD11b-FITC, HLA DR-FITC, IgG-PE, or IgG-FITC. Data were then collected by BD Accuri C6 Plus and analyzed using FlowJo software.

[0030] 1.3, CD146 + / CD146 － MSC purification Human mesenchymal stem cells (MSCs) extracted from umbilical cords and expanded using DMEM medium were prepared for cell purification at passage 3. These MSCs were dissociated by TrypLE and stained with anti-CD146-PE or IgG-PE antibodies. Subsequently, CD146 + MSC and CD146 － MSC were purified.

[0031] 1.4, ELISA and qPCR experiments In ELISA and qPCR experiments, CD146 + MSC or CD146 － MSC were seeded onto 12-well plates at 20 x 10 4Cell culture supernatants were collected after three days. The protein levels of CCL2 and TSG6 were determined using CCL2 and TSG6 Human MCP-1 / CCL2 ELISA Kit (Sigma) and Human TSG6 ELISA Kit (Thermo Fisher Scientific), respectively, according to the manufacturer's instructions.

[0032] Peripheral blood was collected from the eyes of mice (C57BL / 6J, female, 8 weeks old), and the levels of IL-6, TNF-a, IFN-g, and IL-1b in the serum were determined using ELISA kits (BioLegend) according to the manufacturer's instructions.

[0033] 1.5, MSC-PBMC co-culture experiment Whole blood samples were collected from 8 healthy subjects in 10 mL EDTA tubes. All donors signed a written informed consent before the study. The study was approved by the Ethics Committee of the Second People's Hospital of Shenzhen and followed the principles of the Declaration of Helsinki. Human peripheral blood mononuclear cells (PBMCs) were purified using EasySep™ Direct Human PBMC Isolation Kit, followed by MSC-PBMC co-culture experiments. Specifically, PBMCs were stimulated with Dynabeads® Human T-Activator CD3 / CD28 for 24 hours, and then co-cultured with purified CD146 + MSC or CD146 － MSC (20 x 10 4 PBMCs were co-incubated with 5 x 10 4 MSCs in a 37°C, 5% CO2 incubator for 72 hours. Cell proliferation was then evaluated by Cell proliferation Kit I (Roche) and quantified using a Bio-Rad automated microplate reader at a wavelength of 570 nm.

[0034] 1.6, Cell proliferation analysis The evaluation method of cell proliferation was as follows: CD146 + MSC or CD146 － MSCs were seeded in six-well cell culture plates at a density of 10 x 10 4 cells per well. When the confluence reached 80-90%, MSCs were dissociated using TrypLE enzyme. The number of cells was counted using a hemocytometer, and dead cells were identified using a cytotoxicity detection kit (Sigma).

[0035] 1.7, Mouse model and treatment (1) Acute inflammation mouse model and T2D diabetic mouse model Mice (C57BL / 6J, female, 8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center and were raised in a specific pathogen-free (SPF) environment. This study followed the ARRIVE guidelines and has been approved by the Animal Research Ethics Committee of Shenzhen Second People's Hospital. The mice were divided into 3 groups, 8 in each group, as follows: Group 1, PBS transplanted mice; Group 2, CD146 + MSC transplanted mice; Group 3, CD146 － MSC transplanted mice, an acute inflammation model was induced by endotoxin LPS (lipopolysaccharide). Briefly, LPS was injected intraperitoneally into mice at a dose of 20 mg / kg. Ten minutes later, PBS, CD146 + MSC or CD146 － MSC was injected intraperitoneally into the mouse model at a dose of 1 x 10 6 cells per mouse.

[0036] Mice (C57BL / 6J, female, 8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center and were raised in a specific pathogen-free (SPF) environment. This study followed the ARRIVE guidelines and has been approved by the Animal Research Ethics Committee of Shenzhen Second People's Hospital. The T2D mouse model was induced by STZ, with 60 mg / kg STZ injected intraperitoneally every day for five consecutive days. When the fasting blood glucose level of the mouse was ≥16.7 mmol / L, it was confirmed to have type 2 diabetes (T2D). The mice were divided into 4 groups, 8 in each group, as follows: Group 1, normal mice transplanted with PBS (Control+PBS); Group 2, T2D mice transplanted with PBS (T2D+PBS); Group 3, T2D mice transplanted with MSC (T2D+MSC); Group 4, T2D mice transplanted with CD146 + MSC (T2D+CD146 + MSC). The mice in each group were treated twice a week for 6 consecutive weeks by injecting 200 μL of PBS solution containing MSC or CD146 + MSC through the tail vein. The total number of cells in the MSC or CD146 + MSC group was about 10 5 .

[0037] (2) Metabolic parameter analysis MSC or CD146 +Body weight and random blood glucose levels were monitored weekly from the start of MSC treatment until two weeks after the end of the intervention. Intra-peritoneal glucose tolerance test (IPGTT, 1.5 g / kg) and intra-peritoneal insulin tolerance test (IPITT, 0.75 U / kg) were performed and serum insulin levels were measured 26 times by collecting blood samples from the angular vein before and 30 days after the start of the IPGTT. Blood glucose levels were measured at the tip of the tail using AccuChek® Performa.

[0038] 1.8 Glucose-stimulated insulin secretion (GSIS) One mL of INS-1 cell suspension (10 6 cells) without glucose was incubated in 10 mL of KRBS-Ringer bicarbonate solution containing 2.5 mmol / L glucose for 1 hour at 37°C and the resulting supernatant was collected. Subsequently, one mL of INS-1 cell suspension (10 6 cells) without glucose was incubated in 10 mL of KRBS solution containing 25 mmol / L glucose for another 1 hour and the supernatant was further collected. The insulin release under stimulation was quantified by measuring the insulin level in the collected supernatant using a kit (Blue Gene, Shanghai, China). Finally, the insulin level was normalized to the cell protein concentration.

[0039] 1.9 Hematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) staining HE staining was performed using a hematoxylin and eosin staining kit (Beyotime, China) according to the manufacturer's instructions. For IHC staining, the sections were first deparaffinized and then subjected to antigen retrieval with citrate buffer. To inhibit endogenous peroxidase and avoid non-specific binding, a 3% hydrogen peroxide solution was used for 15 min, followed by incubation with goat serum for 30 min and incubation with the primary antibody (GK600505, China) overnight at 4°C. The next day, the secondary antibody (ZSGB-Bio, China) and DAB staining were performed according to the IHC kit instructions. For IF staining, the antigen was first recovered and non-specific sites were blocked, then the antibody (GK600505, China) was used for staining. The next day, a fluorescein-labeled secondary antibody (ZSGB-Bio, China) was applied for 1 hour at 37°C, followed by DAPI staining, and fluorescent microscopic images were taken using a fluorescence microscope.

[0040] 1.10 Intracellularization analysis In vitro studies were conducted using DIO (Invitrogen, V22886, USA) cell labeling solution, and in vivo studies were conducted using the infrared fluorescent DIR (Invitrogen, D12731, USA) lipid-soluble dye. The migration efficiency of conventional MIN-6 cells was assessed by flow cytometry. Fluorescence imaging techniques were used to evaluate MSCs and 146 cells in the pancreas of live mice. + MSC cell migration. The staining procedure was strictly followed according to the manufacturer's instructions.

[0041] 1.11 Biodistribution Imaging MSCs were labeled with DIR (Invitrogen, D12731, USA) and 146 μg of DIR (Invitrogen, D12731, USA) were injected via the tail vein. + MSC cells. Whole-body imaging was performed at 2, 6, and 12 hours, and fluorescence imaging of anatomical organs (such as pancreas, liver, spleen, lung, kidney, and heart) was performed at 12 hours. Simultaneously, fluorescence signals were monitored by collecting blood samples from the inner canthus at 1, 3, and 6 hours, and images were acquired using a Tanon ABL-X5 in vivo fluorescence tomography system.

[0042] 1.12 Statistical Data Statistical data are expressed as mean ± standard error (SEM) and analyzed using SPSS software. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups when the data were normally distributed, with parametric tests and Tukey's post-hoc test performed. P < 0.05 was considered statistically significant.

[0043] 2. Experimental Results 2.1, CD146 + MSCs have a stronger differentiation ability. To further study CD146 + To investigate the function of MSCs, we purified them using fluorescence-activated cell sorting (FACS) technology. The results showed that CD146... + MSC and CD146 － MSCs are similar in morphology ( Figure 1 A), and they are also similar in expression levels of MSC markers. Immunofluorescence analysis showed that CD146 + CD146 expression was detected in MSCs, and CD146... － It was not detected in the MSC. Figure 1 B). Tridifferentiation experiments showed that CD146 + MSCs differentiate into adipocytes ( Figure 1 C, D), bone cells ( Figure 1 E, F) and chondrocytes (Figure 1 G–J) is more efficient. Furthermore, CD146 + MSCs proliferate faster than CD146 － MSC slow ( Figure 1 K). Therefore, CD146 + MSCs not only possess typical MSC characteristics, but also exhibit better tridifferentiation efficiency and a slower proliferation rate.

[0044] 2.2, CD146 + MSCs exhibited significantly stronger inhibitory activity. Subsequently, CD146 was evaluated using an MSC-PBMC co-culture experiment. + MSC and CD146 － Immunosuppressive effects between MSCs. Results showed that both types of mesenchymal stem cells exhibited similar levels of inhibition of lymphocyte proliferation. Figure 2 A). However, in IFN-γ simulation experiments, CD146 + MSCs showed significantly stronger inhibitory activity. Figure 2 A). To further verify CD146 + To investigate the immunosuppressive capacity of MSCs in vivo, we established an LPS-induced acute inflammation model in mice. Indeed, CD146... + MSC maintained its organizational structure ( Figure 2 B), and more than CD146 － MSCs significantly reduced CD45. + Lymphocyte infiltration ( Figure 2 C). In addition, CD146 + MSCs also significantly reduced neutrophil infiltration. Figure 2 D, E) and serum levels of pro-inflammatory cytokines (IL-6, TNF-α, IFN-γ and IL-1β) Figure 2 F). Therefore, CD146 + MSCs exhibit stronger immunosuppressive effects both in vitro and in vivo.

[0045] 2.3, CD146 + MSCs enhanced β-cell targeting and prolonged circulation time. To further study CD146 + The localization of MSCs in pancreatic β cells was investigated, and CD146 was studied. + The tissue distribution of MSCs in vivo was investigated, and the effect of β cells on CD146 was studied. + In vitro migration rate of MSCs. DIR-labeled MSCs and CD146 were injected via tail vein. + Whole-body imaging of mice was performed at 2, 6, and 12 hours after MSC administration.Figure 3 A). At 12 hours, organs were dissected and fluorescence imaging was performed. Compared with the MSC group, CD146 + The pancreatic fluorescence intensity was significantly higher in the MSC-treated group. Figure 3 (B and 3C). In addition, CD146 + MSCs can improve blood circulation time. Therefore, we used DIR-labeled CD146 at 1.3 and 6 hours post-treatment. + MSC and MSC detection of fluorescence signals in blood showed that CD146 + The blood fluorescence signal in the MSC-treated group was significantly higher than that in the MSC-treated group. Figure 3 (D and 3E). For fluorescence imaging of other organs, such as... Figure 3 As shown in F, CD146 + The liver fluorescence signal after MSC treatment was also stronger than that after CD146 treatment. + MSC treatment resulted in richer fluorescence signals in the blood. Compared to MIN-6 cells treated with MSCs, CD146 cells showed significantly higher fluorescence intensity. + MSC-treated MIN-6 cells showed higher migration levels. Figure 3 G-3I). These results indicate that CD146 + MSCs have the ability to target pancreatic β cells and have a longer blood circulation time.

[0046] 2.4, CD146 + MSCs showed stronger β-cell function and improved number compared to MSCs. To investigate CD146 + Whether MSCs have a stronger islet protection effect than MSCs was investigated using a T2D mouse model. Figure 4 A). Although the weight status of the mice in the two groups was similar, CD146 + MSCs are more effective than MSCs in lowering random blood glucose levels. Figure 4 B and 4C). Accept CD146 + MSC-treated T2D mice showed significantly improved glucose tolerance and insulin secretion at 30 min after fasting and during the IPGTT. Figure 4 Mice treated with standard MSCs (D and 4E) showed no significant improvement, while those treated with CD146 showed no improvement. + There was no significant difference in insulin sensitivity between MSCs. Figure 4 F and 4G). This finding is related to CD146. + The fact that MSCs specifically target pancreatic β cells is consistent with ( Figure 4H), since insulin sensitivity reflects the level of insulin resistance in peripheral organs. HE staining and IF staining analysis showed that CD146 + MSC treatment significantly increased the islet area, beta cell area, and the proportion of insulin-positive cells Figure 4 I to 4M. These results indicate that the improvement in beta cell function and number by CD146 + MSCs enhanced the improvement in beta cell function and number.

[0047] The above detailed description of the embodiments of the present application is not intended to limit the present application. Various changes, modifications, replacements, and variations of the embodiments can be made by those skilled in the art without departing from the principles and spirit of the present application, and still fall within the scope of the present application.

Claims

1. CD146 + Application of MSC cells in the preparation of drugs for treating type 2 diabetes.

2. The application according to claim 1, characterized in that, CD146 + The preparation method of MSC cells is as follows: human mesenchymal stem cells (MSCs) are first extracted from the umbilical cord and expanded in DMEM medium containing 5% human platelet cell lysis buffer. After passage, they are stained with anti-CD146-PE or IgG-PE antibodies and finally purified using a cell sorting instrument.

3. The application according to claim 2, characterized in that, MSC was used for CD146 after being passaged to the 3rd generation. + MSC cell purification.

4. The application according to claim 2, characterized in that, Before antibody staining, TrypLE was used to dissociate MSCs.

5. The application according to claim 2, characterized in that, Human umbilical cord was cut into pieces, digested with 1-2 mg / mL collagenase B, and then amplified in DMEM medium containing 5% human platelet cell lysate to obtain MSCs.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, antioxidants, adsorbents, filter aids, and release inhibitors.

8. The application according to claim 6, characterized in that, The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

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