Preparation of CD317 + MSC cell and application of CD317 + MSC cell in treatment of type 2 diabetes

Abstract

The invention belongs to the technical field of medicines, and particularly relates to preparation of CD317 + MSC cells and application of the CD317 + MSC cells to treatment of type 2 diabetes mellitus. The invention researches the characteristics of CD317 + cells in a heterogeneous UC-MSC population, and finds that the FACS is more beneficial to screening of CD317 + MSCs, and the number of the CD317 + MSCs is gradually increased in in-vitro culture. The expression quantity of stem cell characteristic-related genes in the CD317 + MSC subgroup is obviously increased, which indicates that the stem cell is possibly in an undifferentiated state or affects the differentiation potential, but the effect is transiently and gradually weakened along with culture, which explains that the pluripotent differentiation potential and the like are not obviously changed and are not associated with other potential stem cell surface antigens. The research also finds that the cell is a more favorable subgroup, has unique pluripotency and repair / substitution characteristics, can realize stem cell rejuvenation phenotype, can improve pancreas beta cell functions, reduces diabetic complications, enables partial patients to be independent of insulin, and improves the treatment effect on type 2 diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to CD317 + Preparation of MSC cells and application thereof in treating type 2 diabetes. BACKGROUND

[0002] Type 2 diabetes (T2D) is characterized by impaired insulin sensitivity in insulin target tissues and dysfunction and death of pancreatic beta cells. Among them, liver, adipose tissue and skeletal muscle are the three major insulin target tissues, which bear the function of insulin metabolism and play a key role in maintaining blood glucose homeostasis. Inflammation can impair insulin sensitivity and exacerbate diabetic complications, including retinopathy, neuropathy and vasculopathy. Proinflammatory cytokines produced by macrophages and other innate immune cells, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1b), can negatively affect insulin sensitivity. In addition, beta cell dysfunction and death are another major feature of type 2 diabetes, usually occurring within a few years after the onset of diabetes. Currently, type 2 diabetes patients need to rely on insulin sensitizers or exogenous insulin injections to maintain blood glucose within the normal range. However, these treatment methods not only have a heavy burden, but also cannot completely prevent the progression of diabetic complications. Therefore, if a more optimal treatment plan can be developed to improve insulin sensitivity and promote the survival and function of pancreatic beta cells, it will bring good news to type 2 diabetes (T2D) patients.

[0003] In recent years, studies have shown that stem cells have the functions of self-renewal, differentiation into other cell types and immune regulation. Cell-based therapy, with its unique advantages, opens up a new way for the treatment of type 2 diabetes (T2D) - it can provide a continuous source of insulin-producing cells, repair beta cell function, regulate metabolic levels, and improve immune function. However, due to the variety of stem cells and significant differences in efficacy and source, their therapeutic effects and application ranges are also different.

[0004] Mesenchymal stem cells (MSCs) are a type of multipotent adult cells that can differentiate into mesodermal tissues such as osteocytes, chondrocytes and adipocytes. However, many research teams have revealed the broader differentiation potential of mesenchymal stem cells by proposing methods to obtain neurons, hepatocytes and other cells. Some researchers have even further pointed out that mesenchymal stem cells may exhibit characteristics similar to pluripotent cells by expressing transcription factors such as Sox2, Nanog, Oct4 and other stem cell-related transcription factors, and thus can differentiate into all cells of the three germ layers. Although these observations are still controversial, the differences in conclusions between different research teams can be explained by the heterogeneity of mesenchymal stem cells.

[0005] The heterogeneity of MSCs presents a major challenge for further research, as only a small fraction of cells in a MSC population meet the functional criteria for stem cells. The presence of surface antigens associated with other cell types is one manifestation of the observed heterogeneity of MSCs. In reference to the research findings of induced pluripotent stem cells (iPSCs) and embryonic stem cells, researchers have proposed several potential "true stem cell populations" aimed at improving the efficiency of MSC therapy. Among them, SSEA-3 positive cells (a type of early embryonic antigen positive cell) have been proven to be able to differentiate into all cells of the three germ layers. However, the proportion of such cells in the initial cell population is extremely low, which is not consistent with the plasticity characteristics of MSCs. CD271 positive cells (a typical neural crest-derived cell antigen positive cell) have a faster proliferation rate and a higher number of cells that can form colonies. MSCs expressing CD146 exhibit stronger ability to migrate to damaged tissues. CD133 (a surface antigen associated with glioblastoma cells) is also considered as a potential marker for stem cell populations in UC-MSCs and AD-MSCs. In addition, stage-specific embryonic antigen-4 (SSEA-4) is another marker that exists in induced pluripotent stem cells (iPS cells) and embryonic stem cells (ESCs), and also has a high expression rate in MSCs.

[0006] CD317, also known as bone marrow stromal cell antigen 2 (BST-2), tetherin or HM1.24, is an integral membrane protein with a novel topology that is widely expressed in various cells. It was first discovered as a surface marker of late-differentiated and tumor B cells, and later found to have multiple cellular functions. CD317 is widely expressed in various cells, including hepatocytes, alveolar wall cells, activated T cells, monocytes, plasma dendritic cells, pancreatic cells, kidney cells, and vascular endothelial cells. Transcriptome analysis shows that CD317 is also expressed in human and mouse platelets. CD317 is associated with many biological processes, including limiting the release and replication of enveloped viruses, regulating B cell growth, and stabilizing membrane microdomains. However, no studies have shown that CD317 is related to the treatment of type 2 diabetes. + . SUMMARY

[0007] To overcome the shortcomings of the prior art, the present application explores the role of CD317 + The therapeutic potential of MSCs as a potential pluripotent stem cell population with "mesenchymal stem cell plasticity" is to determine their characteristics and application value in the treatment of type 2 diabetes.

[0008] To achieve the above object, the technical scheme adopted by the present application is: The present application provides CD317 + The application of MSC cells in the preparation of a drug for treating type 2 diabetes.

[0009] Preferably, the CD317 + The MSC cells are derived from umbilical cord mesenchymal stem cells (UC-MSC). Compared with adipose tissue mesenchymal stem cells (AD-MSC), CD317 + The number of cells is more, which is 3-4 times of AD-MSC.

[0010] More preferably, the CD317 + The preparation method of the MSC cells is as follows: the umbilical cord is made into a Wharton's jelly segment, and then placed in DMEM for culture, the DMEM contains 4-5% human platelet cell lysate, and penicillin-streptomycin-amphotericin B, the dilution ratio of the penicillin, streptomycin and amphotericin B is all 1:100; when the cells are cultured to complete fusion and start to migrate from the Wharton's jelly segment, the cells are dissociated using Accutase cell dissociation solution, and then umbilical cord mesenchymal stem cells (UC-MSC) are obtained by flow sorting, and CD317 + MSC cells are obtained by cell sorting.

[0011] Further, the human platelet cell lysate is PLTGold clinical grade human platelet lysate. The PLTGold human platelet lysate belongs to clinical grade human platelet lysate, and can increase the proportion of positive cells to about 70%.

[0012] Further, the cell sorting adopts a FACS sorting method. The FACS sorting method shows better cell recovery rate and lower mortality rate.

[0013] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0014] Still further, the excipient comprises at least one of an excipient, a propellant, a solubilizer, a cosolvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a clathrate agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, an antioxidant, an adsorbent, a filter aid, a release retardant.

[0015] Furthermore, the dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention studies CD317 isolated from heterogeneous UC-MSC populations. + Cell characteristics, depending on different culture conditions, CD317 in UC-MSCs + The cell proportion is approximately between 35% and 70%. An environment rich in protein and nutrients is more conducive to CD317. ＋ The enrichment of cells is likely due to the fact that these conditions provide the necessary matrix for the synthesis of this type of cell. A comparison of magnetically activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) revealed that flow cytometry (FACS) is more effective in screening for positive CD317 cells. + MSCs, their numbers gradually increased during further in vitro culture. Further research revealed that CD317... + The relative expression levels of genes associated with stem cell characteristics were significantly elevated in the MSC subset, suggesting that this subset may be in an undifferentiated state, which could potentially affect its differentiation potential. However, this effect is transient and gradually weakens with continued cell culture. This phenomenon may explain why, in CD317... + In the MSC-positive population, there were no significant changes in pluripotent differentiation potential, proliferation rate, or colony-forming ability. Furthermore, no CD317 was found in the study. + The MSC population is associated with other potential stem cell surface antigens. This invention also found that CD317... + MSC cells may be a more favorable subset due to their unique pluripotency and repair / replacement properties, enabling the realization of a younger mesenchymal stem cell phenotype. The final study showed that CD317... + MSCs not only improve the function of pancreatic β cells, but also reduce the incidence of diabetic complications, enabling some patients to achieve insulin independence, thereby improving the therapeutic effect of these high-quality mesenchymal stem cells on type 2 diabetes. Attached Figure Description

[0017] Figure 1 CD317 in mesenchymal stem cells (MSCs) + Figures showing experimental results of cell-related characteristics studies; A: Immunofluorescence staining graph; B, C: Flow cytometry analysis and statistical bar charts; D: Bar chart; E, F: Bar charts and flow cytometry analysis graphs.

[0018] Figure 2 CD317 from UC-MSC+ MSCs with different properties; A, B, C are column charts; D is a flow cytometry histogram.

[0019] Figure 3 Statistical charts of experimental results for UC-MSC sorting and related property verification; A, B, C: flow cytometry analysis charts; D: immunocytochemical staining chart.

[0020] Figure 4 Experimental result charts for surface antigen expression analysis of UC-MSC after sorting; A, C, E, G: flow cytometry histograms; B, D, F, H: column charts; I: scatter plot.

[0021] Figure 5 Column charts of experimental results for secretion profile analysis of UC-MSC at different time points after sorting; A: secretion profile at 3 days after FACS sorting; B: secretion profile at 5 days after FACS sorting.

[0022] Figure 6 CD317 + Statistical charts of experimental results of MSC intervention in a type 2 diabetes (T2D) mouse model; A: flowchart; B, C, D, F: line charts; E, G, H, L: column charts; I, M: box plots; J: pancreatic tissue histological staining (such as H&E staining); K: islet cell immunofluorescence staining (insulin, glucagon, DAPI nuclear staining). DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each of the embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0024] 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.

[0025] The present application aims to explore the therapeutic potential of CD317 positive subpopulation (CD317 + MSC) of umbilical cord-derived mesenchymal stem cells (UC-MSC) as a potential pluripotent stem cell population with "mesenchymal stem cell plasticity". Such cells have the ability to repair damaged cells. To clarify the unique properties of the CD317 + subpopulation, it was studied in comparison with the CD317 negative subpopulation (CD317 -and unclassified umbilical cord mesenchymal stem cells (UC-MSC) heterogeneous population. Through experiments, the best preparation conditions for UC-MSC expression and separation were determined, and the expression of CD317 + Analysis of MSC positive subpopulation also revealed comprehensive features related to its stem cell properties. Further studies found that CD317 + MSC can play an insulin sensitizing role by restoring glucose uptake under insulin stimulation. It not only improves hyperglycemic state, improves glucose tolerance and metabolic balance, but also protects the number of pancreatic beta cells and promotes their proliferation.

[0026] In order to comprehensively and clearly present the technical solutions of the present application and its significant advantages, the present application will be further elaborated in detail below.

[0027] 1. Experimental method 1.1, Separation and primary culture of UC-MSC According to the guidelines of the Ethics Committee of the Second People's Hospital of Shenzhen, after obtaining the mother's written consent, the length of the human umbilical cord was obtained from the full-term delivery of the baby. First, the obtained umbilical cord was transported back to the laboratory in phosphate buffered saline (PBS) containing 1% penicillin-streptomycin-amphotericin B. Upon arrival at the laboratory, the umbilical cord was cut into 2 to 3 mm thick slices using a needle, and then 2-3 mm diameter cylindrical Wharton's jelly fragments were obtained using a biopsy punch with a diameter.

[0028] Next, these Wharton's jelly fragments were transferred to 6-well culture plates and cultured under standard conditions for UC-MSC culture. The culture medium used was DMEM containing 5% human platelet lysate (including PLT Gold clinical grade from Mill Creek Life Sciences, MultiPL30 from Macopharma and MultiPL100 from Macopharma), and penicillin-streptomycin-amphotericin B (dilution ratio 1:100); cell culture was performed using adherent surface culture, and the environmental conditions were 37°C temperature, 95% humidity, 5% CO2 and 5% O2 concentration, and the culture medium was replaced every two days, and the culture was continued for 14 days. During the culture process, the expression of CD317 in the culture population was compared using different human platelet lysates, and PLT Gold clinical grade was selected as the lysate for subsequent experiments.

[0029] When the cells were cultured to complete confluence and began to migrate from the fragments of Wharton's jelly, the cells were dissociated using Accutase cell dissociation solution (Beckton Dickinson, Franklin Lakes, NJ, USA), followed by counting.

[0030] 1.2, Flow cytometry After the cells were dissociated by Accutase cell dissociation solution, they were washed with PBS, then resuspended in PBS buffer, and then subjected to flow sorting. When flow cytometry analysis was performed, the corresponding antibodies (Table 1) were used, and the cells were incubated with the diluted antibodies in the dark for 30 minutes. After the incubation, the cells were washed twice with staining buffer, resuspended in PBS buffer again, and the obtained UC-MSC (obtained by CD317 and its isotype control) were finally analyzed using FACS Canto II, FACSDiva software and FlowJo 10.

[0031] Table 1 Antibodies used for flow cytometry analysis 1.3, Culture of adipose tissue mesenchymal stem cells (AD-MSC) The AD-MSC (AD-MSC seed cells donated by the Second People's Hospital of Shenzhen) were placed in MEMa medium containing 5% human platelet lysate and 1% penicillin-streptomycin-amphotericin B (at a ratio of 1:100 with the culture medium). The cells were cultured by adherent culture, and the culture conditions were 37°C temperature, 95% humidity, 5% CO2 and 5% O2 concentration, and the culture medium was replaced every 2-3 days. When the AD-MSC reached a semi-confluent state, they were passaged, and the third passage AD-MSC were dissociated by Accutase cell dissociation solution and washed twice with PBS, then treated according to the preparation method of the flow cytometry analysis described above for flow cytometry analysis.

[0032] 1.4, Magnetic activated cell sorting (MACS) separation of CD317 + MSC cells From the third passage of UC-MSC, CD317 + MSC cells were separated using MACS separation technology. The specific steps are as follows: first, the UC-MSC were dissociated and counted; then, about 2-10 x 10 6Cells were incubated with magnetic beads in the anti-CD317 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 minutes in the dark; after that, cells were washed with PBS; then, cells were resuspended in a PBS solution containing 1% BSA and loaded into an automatic MACS Pro separator, cells were run in a magnetic field by Possel S device. For further analysis, the population of cells that passed through the column was retained and collected as a second fraction. After MACS sorting was completed, cells were counted on a hemocytometer using trypan blue to calculate total cell number and cell viability. Finally, cells were recultivated for subsequent experimental studies.

[0033] 1.5. Fluorescence-activated cell sorting (FACS) separation of CD317 + MSC cells Third passage UC-MSC were taken for FACS separation of CD317 + MSCs were stained according to flow cytometry staining procedures (CD317 fluorescent antibody (BD) was mixed with cells and incubated for 2 hours, cells were washed with PBS), and sorted using a FACS Aria IIu flow cytometry laboratory. The laser configuration used for sorting was: violet - 407 nm (detector: 450 / 40, 530 / 30), blue - 488 nm (detector: 488 / 10, 530 / 30, 585 / 42, 616 / 23, 695 / 40, 780 / 60), red - 633 nm.

[0034] CD317 - and CD317 + Cells were collected from both populations and resuspended in cell culture medium (DMEM containing 5% human platelet lysate, and penicillin-streptomycin-amphotericin B (dilution ratio 1:100)). Immediately after FACS sorting, the obtained populations were analyzed again using FACS Aria to confirm the sorting purity. Subsequently, the total cell number and viability were counted using a hemocytometer and trypan blue. After that, cells were seeded in culture dishes for further experimental studies.

[0035] 1.6. Optimization of cell sorting parameters To determine the CD317 + More efficient separation of MSC cells, the recovery rate, viability, yield, and purity of MACS and FACS sorting were compared. The recovery rate refers to the ratio of the number of cells obtained in the forward fraction to the number of cells used for sorting, calculated by counting the cells in the fraction before and after sorting. Viability was described by checking cell death rate in samples after cell isolation by trypan blue staining. Yield was the ratio of forward cell content before and after cell isolation, estimated by flow cytometry analysis of samples. Purity refers to the percentage of CD317 + Percentage of MSC cells.

[0036] 1.7. Immunocytochemistry To detect unsorted umbilical cord mesenchymal stem cells (UC-MSC), unsorted umbilical cord mesenchymal stem cells CD317 - MSC and unsorted umbilical cord mesenchymal stem cells CD317 + MSC, immunocytochemistry was performed following these steps: first, UC-MSC were washed with PBS and then fixed in 4% paraformaldehyde for 15 min; next, samples were incubated with blocking mixture containing 10% goat serum and 1% bovine serum albumin for 1 h at room temperature; then, primary antibodies (Table 2) were incubated for 24 h at 4°C; the next day, cells were washed with PBS and incubated with fluorochrome-labeled secondary antibodies (Table 2) for 1 h; finally, samples were mounted with Fluoromount-G containing DAPI (DAPI was used to stain the cell nuclei). The whole analysis was performed on a Zeiss confocal microscope.

[0037] Table 2. Antibodies used for immunocytochemistry analysis 1.8. Soluble secretome analysis The soluble secretome of unsorted, positive and negative cell populations was analyzed using the human magnetic Luminex assay (R&D Systems, Minneapolis, MN, United States). The procedure was as follows: FACS-sorted cells were seeded at a density of 2,000 cells per square centimeter; at 3 and 5 days after FACS sorting, cell culture medium was collected, while standard medium was used as a negative control. The molecules detected included: epithelial growth factor (EGF), basic fibroblast growth factor (bFGF), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), chemokine ligand 2 (CCL2), leukemia inhibitory factor (LIF), angiogenin, vascular endothelial growth factor-c (VEGF-c) and intercellular adhesion molecule 1 (ICAM-1). Actual levels of secreted factors were determined by subtracting the negative control values from the results obtained. Luminex assays were performed to measure the levels of secreted factors in the supernatants of the cells.

[0038] 1.9. Animal model and treatment Five-week-old male C57BL / 6J mice were selected and after an adaptation period of 1-2 weeks, the mice were grouped and treated, with 18 mice in each group: one group received a high-fat diet (HFD) regimen for 12 weeks, with a fat content of 60%; the other group served as a control group and received a normal chow diet. Subsequently, the HFD group of mice was intraperitoneally injected with 60 mg / kg STZ for five consecutive days, starting on day 1. When the fasting blood glucose level of the mice was ≥ 16.7 mmol / L, it was confirmed that they had type 2 diabetes (T2D). To study the effect of CD317 + MSC on islet function and its underlying mechanisms, the mice were divided into three groups, with 6 mice in each group: Control + PBS group, T2D + PBS group, and T2D + CD317 + MSC group. The mice in each group were treated twice a week for 6 consecutive weeks by tail vein injection of 200 μL of a PBS solution, in which the T2D + CD317 + MSC group was injected with a PBS solution containing CD317 + MSC, with a total cell number of about 10 5 The animal experiment protocol was approved by the Animal Ethics Committee of the Second People's Hospital of Shenzhen, Sun Yat-sen University, and was strictly conducted in accordance with the guidelines established by the National Institutes of Health (NIH) of the United States.

[0039] 1.10. Metabolic parameter analysis From the start of MSC or CD146 + MSC treatment until two weeks after the end of the intervention, body weight and random blood glucose levels were monitored weekly. At the same time, intraperitoneal glucose tolerance tests (IPGTT, 1.5 g / kg) and intraperitoneal insulin tolerance tests (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 also measured at the tip of the tail using AccuChek® Performa.

[0040] 1.11. Glucose-stimulated insulin secretion (GSIS) A 1 mL suspension of INS-1 cells lacking glucose (with a cell number of 10 6(1) 10 mL KRBS-Ringer bicarbonate solution containing 2.5 mmol / L glucose was added, and the cells were incubated at 37 °C for 1 h, and the supernatant was collected. Subsequently, 1 mL of INS-1 cell suspension (cell number 10 6 (2) 10 mL KRBS solution containing 25 mmol / L glucose was added, and the cells were incubated at 37 °C for 1 h, 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.

[0041] 1.12. Statistical analysis The normality of the data was checked using the Shapiro-Wilk normality test. For two groups of data, if normally distributed, the unpaired t-test was used; if multiple groups of data were normally distributed, one-way analysis of variance (ANOVA) was first performed, and then Tuckey's multiple comparison test was performed. For non-normally distributed data, Kruskal-Wallis test was first performed, and then Dunn's multiple comparison test was performed. The statistical results were expressed as mean ± standard deviation (SD) for parametric test results, or median ± 95% confidence interval (95% CI) for non-parametric test results. When the p-value was greater than 0.05, the results were considered statistically significant. Statistical analysis was completed using GraphPad Prism 7 software.

[0042] 2. Experimental results 2.1. Expression of CD317 in heterogeneous UC-MSC population The results are shown in FIG. 1, where A shows CD317 (green), CD90 (red), and cell nucleus DAPI (blue) staining in UC-MSC, verifying that CD317 Figure 1 MSC expresses CD90 (mesenchymal stem cell recommended antigen); B, C compares the proportion of CD317 + cells in UC-MSC and AD-MSC, with a higher proportion in UC-MSC; D presents the proportion of CD317 + cells during the passage of UC-MSC (passage 1, 3, 5), with no significant change; E, F compares the proportion of CD317 + cells under the culture of three kinds of platelet lysates (MultiLP30, MultiLP100, PLTGold), and MultiLP30 is selected to facilitate the expression of CD317 + cells; G, H compares the proportion of CD317 +The culture medium of MSC growth was determined for subsequent experiments to identify the cell and culture conditions. The results showed that the UC-MSC used for the experiment exhibited the characteristics of mesenchymal stem cells (MSC) recommended by the International Society for Cellular and Gene Therapy. All CD317 + MSC cells expressed CD90, which is one of the recommended mesenchymal stem cell antigens Figure 1 (A). To determine the most favorable conditions, this study evaluated the source of CD317 + cells, the number of passages, and the culture medium in heterogeneous mesenchymal stem cells (MSC). This study compared the CD317 + content of MSC cells in two different populations of mesenchymal stem cells (MSC) from different tissues: umbilical cord mesenchymal stem cells (UC-MSC) and adipose tissue mesenchymal stem cells (AD-MSC) (B, C in Figure 1 ). The results showed that the number of CD317 + cells in UC-MSC was almost 3.5 times higher than that in AD-MSC (70% ± 8.3% and 20.3% ± 11.7%, respectively). In addition, the CD317 expression level did not change significantly in the first, third, and fifth passages of UC-MSC cultured in vitro (D in Figure 1 ). To select the optimal culture medium composition, this study compared three commercially available platelet lysates as sources of proteins and chemotactic factors: MultiLP30, MultiLP100, and PLT Gold clinical grade (see E, F in Figure 1 ). The results showed that the growth factor content in MultiLP100 and PLT Gold lysates was higher than that in MultiLP30. The proportion of positive cells was the lowest (35.7% ± 11.1) when cultured with MultiLP30 lysate, while it increased to 74% ± 9.7% and 70% ± 8.3, respectively, when cultured with MultiLP100 and PLT Gold lysates (see F in Figure 1 ). Therefore, from the third passage of UC-MSC, cells cultured with PLT Gold human platelet lysate were selected for further experiments and analysis.

[0043] 2.2 Comparison of CD317 + MSC cell isolation methods To screen a better CD317 + MSC isolation method for UC-MSC, this study compared the MACS and FACS techniques. The comparison indicators included recovery rate (i.e., the ratio of positive cells to the total number of cells used for sorting), survival rate (i.e., cell mortality), yield, and the proportion of positive cells before and after sorting. The results are shown in Figure 2As shown in the figure, A, B, and C represent the differences between MACS and FACS technologies in terms of recovery rate, viability, and yield, respectively; D shows the difference in CD317 expression before and after MACS and FACS sorting. The results showed that the number of cells obtained using FACS technology was more than 13 times that obtained using MACS technology (MACS: 1.6% ± 0.9 vs. FACS: 21.4% ± 7.4). Figure 2 (A) Although cell viability decreased after MACS sorting, this difference was not statistically significant due to the significant difference in MACS sorting results (MACS: 62.75% ± 24.3 vs. FACS: 89% ± 2). Figure 2 (B) The FACS method had a slightly higher yield, but the difference was not statistically significant (MACS: 99.9% ± 11.9 vs. FACS: 148.9% ± 44.4). Figure 2 (C in the text). To calculate the yield, we compared CD317 before and after sorting. + The number of MSC cells ( Figure 2 (D in the text). Overall, we chose the FACS sorting method for subsequent experiments because it showed better cell recovery and lower mortality, and the yield was increasing. Figure 2 The results showed significant differences in CD317 expression in the initial populations, primarily due to the different flow cytometers assigned to this method. MACS sorting was performed using FACS Canto. During FACS sorting, we used the integrated FACS Aria IIu with different detector settings than before (see Materials and Methods section for details). These differences may explain the inconsistency in CD317 expression across different study groups. However, in subsequent experiments, we consistently used the same flow cytometer, e.g., after sorting CD317... + MSC survival status and co-expression of other surface antigens.

[0044] 2.3 CD317 after FACS separation + MSC We obtained two groups of UC-MSC-derived stem cells using FACS, namely a negative subset (CD317). - MSC) and positive subsets (CD317) + MSC), results as follows Figure 3 As shown in the figure. A presents the CD317 expression levels in cells before and after FACS sorting, while B uses a line graph to compare CD317 expression at different passage numbers after sorting. + MSC and CD317 - CD317 in MSC +C uses a histogram to show the expression distribution of CD317 at different passage numbers after FACS; D uses fluorescent labels (CD317 green, CD90 red, DAPI staining blue) to visually represent the expression of MSCs and CD317. - MSC, CD317 + CD317 expression and cell morphology in MSCs were used to verify the CD317 content in the positive population after sorting. + Differences in MSC numbers. Results showed that, to examine purity and yield, cell counts were measured in the undifferentiated population before FACS and the recipient population after FACS. Figure 3 (A) The FACS method makes CD317 in the positive population + The proportion of MSCs reached 87.4% ± 4.3%, while CD317 was present in the negative population. + The proportion of MSCs was 1.2% ± 1.6%. Figure 3 (B) In the following six passages, CD317 was monitored in both positive and negative populations. + MSC content ( Figure 3 (B and C in the original text). In the first four generations, CD317 in both populations... + Significant differences were observed in the percentage of MSCs. CD317 was observed in the negative population. + MSC levels increased. No differences were recorded between the analyzed populations in the sixth passage after FACS. Immunocytochemical staining showed that CD317 levels were higher in the FACS-positive population after FACS isolation. + The number of MSCs has increased significantly. Figure 3 (D in the middle).

[0045] 2.4, CD317 + Expression of other surface antigens in the enriched UC-MSC population We studied CD317 + Whether cell enrichment affected the expression of other surface antigens still present in the mesenchymal stem cell (MSC) population, including CD49F, CD133, CD317, and CD271, was investigated. Results are as follows: Figure 4 As shown. A, C, E, and G represent unsorted MSCs and CD317, respectively. - MSC, CD317 + Expression distribution of CD49F, CD133, Stro-1, and CD271 in MSCs; quantitative statistical analysis of unsorted MSCs and CD317 by B, D, F, and H. - MSC, CD317 +Percentage of MSCs positive for the four corresponding antigens; I shows the expression differences of each antigen in different cell populations as fold change (relative to the unsorted population); Overall description of CD317 + Cell enrichment did not significantly affect the expression of these surface antigens, and CD317 + The MSC population was difficult to associate with other unique subpopulations. Results showed that the percentages of these surface antigens did not change significantly before and after cell sorting. Among the UC-MSC subpopulation, CD49F... + MSCs account for 84% to 94% ( Figure 4 (A, B), CD133 + MSC accounted for 1.4% to 3.5% ( Figure 4 (C, D) Stro-1 + MSC accounted for 74% to 79% ( Figure 4 (E, F) CD271 + MSC accounted for 2.4% to 4% ( Figure 4 (G, H in). For each antigen, the fold change in its expression level is calculated relative to the unsorted cell population (G, H). Figure 4 (I) The most significant changes were observed in antigens that are rare in the population, such as CD133 and CD271. However, we did not find any statistically significant differences, which may be due to significant differences between samples from different donors. Our surface antigen analysis showed that CD317 + MSC populations are almost impossible to link with other distinct subpopulations.

[0046] 2.5 Subgroup Secretion Spectrum In the following steps, we compared unclassified UC-MSCs and CD317. - MSC and CD317 + MSC subsets on day 3 after in vitro FACS sorting ( Figure 5 A) and day 5 Figure 5 The secretion profile of subpopulation B) was analyzed. We measured the levels of selected trophic factors (EGF, bFGF, GDNF, and BDNF), cytokines and chemokines (CCL2 and LIF), and angiogenic factors (angiopoietin, VEGF-c, and ICAM-1) to help investigate changes in secretion characteristics of different subpopulations at different in vitro culture times. Results showed that on day 3 of observation, the secretion of BDNF, HGF, and GDNF increased in the unclassified population. CD317 - The MSC population secreted less VEGF-c on day 3 after FACS sorting. By day 5, its secretion pattern changed slightly. Compared to other variants, CD317... -The MSC population secreted higher levels of bFGF and LIF. Meanwhile, CD317... + The MSC population secreted higher levels of CCL2, but lower levels of VEGF-c.

[0047] 2.6, CD317 + MSCs improve β-cell function and number The results are as follows Figure 6 As shown, A illustrates the experimental procedure, including T2D mouse modeling and CD317... + MSC intervention and detection points; B, C, D, and F respectively show the trends of random blood glucose, weight, glucose tolerance, and insulin sensitivity over time or during the testing process, and compare different groups (Control+PBS, T2D+PBS, T2D+CD317). + The MSC (mesocellular) group showed differences; E, G, H, and L used bar charts to quantitatively analyze glucose metabolism-related indicators (such as area under the blood glucose curve, insulin secretion, etc.) and the proportion of islet-related cells, visually demonstrating the differences between groups; I and M presented the distribution and differences between groups of indicators such as islet area and β-cell area; J presented pancreatic tissue pathological staining (such as H&E staining); K presented islet cell immunofluorescence staining (insulin, glucagon, DAPI staining of nuclei), visually presenting CD317 at the tissue and cellular level. + The effects of MSC intervention on the morphology and distribution of pancreatic and islet cells; used holistically to illustrate CD317 + MSCs can improve the function and number of pancreatic β cells in T2D mice. Results showed that, in order to investigate CD317... + Did MSCs exacerbate pancreatic islet dysfunction? A T2D mouse model was used. Figure 6 (A) As expected, CD317 + Random blood glucose levels decreased significantly after MSC intervention. Figure 6 (B in the text). Additionally, CD317 + MSCs reduced the weight loss in T2D mice. Figure 6 (C) Accept CD317 + MSC-treated T2D mice showed improved glucose tolerance ( Figure 6 D, E), insulin sensitivity ( Figure 6 It showed significant improvement in insulin secretion (F, G) and 30 min after IPGTT (in the context of insulin secretion). Figure 6 (H in the text). In T2D mice, the size of pancreatic islets and pancreatic β cells was significantly reduced compared to normal controls. However, using CD317... + MSC therapy helps reduce the reduction in pancreatic islet and β-cell area. Figure 6I-M). The proportion of insulin-positive cells was lower, while the proportion of glucagon-positive cells was higher in T2D mice. Administration of CD317 + MSC reversed the loss of insulin beta cells Figure 6 J-M). The above data suggest that CD317 + MSC improved the function and number of pancreatic beta cells in T2D mice.

[0048] The above detailed the embodiments of the present application, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments are made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. CD317 + Application of MSC cells in the preparation of drugs for treating type 2 diabetes.

2. The application according to claim 1, characterized in that, The CD317 + MSC cells are derived from umbilical cord mesenchymal stem cells.

3. The application according to claim 2, characterized in that, The CD317 + The method for preparing MSC cells is as follows: Umbilical cord cells are prepared into Wharton's jelly fragments and cultured in DMEM containing 4-5% human platelet cell lysis buffer and penicillin-streptomycin-amphoteric B, with each component diluted 1:

100. When the cells reach complete confluence and begin to migrate from the Wharton's jelly fragments, they are dissociated using Accutase cell dissociation buffer. Umbilical cord mesenchymal stem cells are then obtained by flow cytometry sorting. After further cell sorting, CD317 cells are isolated. + MSC cells.

4. The application according to claim 3, characterized in that, The human platelet cell lysate is PLTGold clinical-grade human platelet lysate.

5. The application according to claim 3, characterized in that, The cells were sorted using the FACS sorting method.

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.

Images