Application of combination of human umbilical cord mesenchymal stem cells and metformin in preparation of anti-DFU drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of human umbilical cord mesenchymal stem cells combined with metformin in preparation of anti-DFU medicines. By constructing a diabetic foot ulcer rat model and combining in-vivo and in-vitro experiments and signal analysis, it is confirmed for the first time that hUC-MSCs and metformin have a synergistic effect in treating diabetic foot ulcer. Combined treatment is beneficial to diabetic foot ulcer wound closure, reepithelization, matrix remodeling and proliferation of blood vessels and inflammation related cells, regulation and control of vascular endothelial cells PI3K-AKT and downstream signal channels thereof can be promoted through paracrine, so that the inhibited survival, proliferation, migration and vascularization capacities of the vascular endothelial cells PI3K-AKT in a hyperglycemia environment are recovered, and the vascular endothelial cells PI3K-AKT and the downstream signal channels of the vascular endothelial cells PI3K-AKT are promoted. Further, the dynamic regulation of enhancement, maturation and weakening of new angiogenesis of the wound of the DFU rat is promoted, and the wound healing is accelerated. The invention provides theoretical basis and experimental support for developing a more effective treatment scheme.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of an anti-DFU drug. Background Art

[0002] Diabetic foot ulcer (DFU) is a chronic wound in which the epidermis and at least part of the dermis of the foot of a diabetic patient ruptures, accompanied by problems such as hyperglycemia, impaired epithelialization, vascular microcirculation disorders, and chronic inflammation. It is one of the serious chronic complications of diabetes. Its overall healing rate is low, and the recurrence rate and amputation rate remain high. It is estimated that approximately 25% of diabetic patients will develop a foot ulcer in their lifetime, and the global epidemiological prevalence of diabetic foot ulcers is 6.3%. Most patients with diabetic foot ulcers usually require surgical intervention for debridement due to recurrent or refractory infection, and even 15% to 20% require amputation for adequate control or healing. The lifetime risk of lower limb amputation in patients with DFU is at least 19%, and the survival rate is significantly worse.

[0003] The healing time and overall healing rate of diabetic foot ulcers vary greatly depending on the cause of the ulcer, size, presence of infection, and patient characteristics. Typically, the wound healing time of patients with DFU ranges from 3 months to more than 12 months, which is significantly longer than the healing time of normal wounds. The treatment of DFU aims to manage early wounds, with the main goal being wound closure. The basic elements are treating the underlying disease process, ensuring adequate blood supply, local wound control (including infection control), and decompression for rapid healing. Conventional treatment of DFU is mainly through medical therapy and surgical intervention to restore blood flow, including wound debridement, hyperbaric oxygen therapy, negative pressure wound therapy, wound dressing, unloading pressure, and ulcer amputation. Although these treatments have certain effects, they are slow to achieve results and are expensive. The amputation and recurrence rates of diabetic patients remain high.

[0004] With the in-depth understanding of the pathological mechanism of diabetic foot ulcers, cytokine drugs and stem cell therapies for wound management and treatment of diabetic foot ulcers have been widely studied. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are considered to be a promising treatment method because of their short doubling time, long survival time, high proliferation ability, strong anti-inflammatory ability, and low expression of HLA-ABC and HLA-DR. Some existing technologies have reported on stem cell preparations for diabetes or its complications. For example, patent CN106110302A discloses a stem cell preparation for the treatment of diabetic foot, including mesenchymal stem cells, low molecular weight heparin sodium, polysaccharides, vitamin B12, methylcobalamin, cephalosporin, vascular endothelial growth factor and solvent, wherein the mesenchymal stem cells are preferably human umbilical cord blood mesenchymal stem cells. For another example, patent CN105477017A discloses a mixed stem cell preparation for the treatment of diabetic foot, which includes placental hematopoietic stem cells and umbilical cord mesenchymal stem cells. This invention introduces placental hematopoietic stem cells into the mesenchymal stem cell solution, which can effectively activate the VEGF signaling pathway. After local injection of the mixed stem cell preparation, it can increase peripheral blood and local VEGF concentrations, promoting the healing of diabetic foot ulcers. However, the survival of stem cells in a long-term hyperglycemic microenvironment may be limited. Therefore, it is necessary to explore the synergistic effects of stem cells combined with other treatment methods to further improve the treatment effect of diabetic foot ulcers.

[0005] Metformin (Met) is a commonly used drug for diabetic patients to regulate blood sugar and improve insulin resistance. It is inexpensive, easy to use, and also has certain biological effects such as inhibiting inflammation, reducing tissue and vascular damage, and antibacterial effects.

[0006] At present, there are no reports on the treatment of diabetic foot ulcers with hUC-MSCs combined with metformin. Summary of the Invention

[0007] In light of this, one of the objectives of the present invention is to provide a method for treating diabetic foot ulcers using human umbilical cord mesenchymal stem cells (hUC-MSCs) in combination with metformin. The present invention demonstrates that the combination of hUC-MSCs and metformin for treating diabetic foot ulcers is more effective than either hUC-MSCs or metformin alone.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a medicament for treating diabetic foot ulcers.

[0010] The present invention focuses on hUC-MSCs and its combined treatment strategy with metformin, and explores the mechanism of action of this treatment strategy in promoting DFU wound healing and angiogenesis in vivo and in vitro. The present invention's study found that the effect of metformin alone on the indicators related to diabetic foot ulcer wound healing and angiogenesis is not completely significant, but metformin can promote the effect of hUC-MSCs in diabetic foot ulcer wound healing and angiogenesis through multiple synergistic mechanisms. First, metformin improves the diabetic conditions of DFU rats, such as hyperglycemia, impaired glucose metabolism, and insulin resistance; secondly, metformin can promote its retention and implantation in diabetic foot ulcer wounds by improving the survival and paracrine function of hUC-MSCs in high-glucose environment wounds; finally, metformin helps the vitality and function of vascular endothelial cells in a high-glycemic environment.

[0011] Preferably, the human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and lowly express CD34, CD45 and HLA-DR.

[0012] Preferably, in the human umbilical cord mesenchymal stem cells, the positive rates of CD73, CD90 and CD105 are ≥95%, and the positive rates of CD34, CD45 and HLA-DR are ≤5%.

[0013] Preferably, the diabetic foot ulcer is accompanied by weight loss, hyperglycemia, impaired glucose metabolism, insulin resistance, delayed wound healing, inflammation, impaired epithelialization, impaired angiogenesis and / or polydipsia and polyuria.

[0014] Preferably, the drug is used to promote wound closure of diabetic foot ulcers;

[0015] and / or the drug is used to promote re-epithelialization and collagen deposition in diabetic foot ulcer wounds;

[0016] and / or the medicament is used to promote regeneration of skin appendages;

[0017] And / or the drug is used to promote scarless wound healing and resist fibrotic scar formation;

[0018] and / or the drug is used to promote wound healing and proliferation;

[0019] And / or the drug is used to inhibit wound inflammation.

[0020] Preferably, the promotion of wound healing proliferation and inhibition of wound inflammation includes promoting the expression of CD31 and CD34, α-SMA, FSP1, K14 and CD163, promoting the expression of VEGFA, TGF-β, bFGF, IL-10, and inhibiting the expression of IL-1β and TNF-α.

[0021] Preferably, the skin appendages include hair follicles.

[0022] Preferably, the promoting of wound collagen deposition includes increasing Col type I, Col III, and the ratio of Col III / type I, and decreasing the expression of MMP2, MMP3, and MMP9.

[0023] A second object of the present invention is to provide a use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of angiogenesis promoters, cell proliferation promoters and / or cell migration promoters for treating diabetic foot ulcers.

[0024] To achieve the above object, the present invention adopts the following technical solutions:

[0025] Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of angiogenesis promoters, cell proliferation promoters and / or cell migration promoters for treating diabetic foot ulcers.

[0026] Preferably, the drug promotes the expression of p-VEGFR2, eNOS, MMP2 and MMP9.

[0027] Preferably, the vascular endothelial cells include HUVEC cells.

[0028] A third object of the present invention is to provide a use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a hypoglycemic drug.

[0029] A fourth object of the present invention is to provide a use of metformin in the preparation of a drug for inhibiting or repairing damage to human umbilical cord mesenchymal stem cells caused by a high sugar environment.

[0030] To achieve the above object, the present invention adopts the following technical solutions:

[0031] Use of metformin in the preparation of a drug for inhibiting or repairing damage to human umbilical cord mesenchymal stem cells caused by a high-glucose environment.

[0032] Preferably, the human umbilical cord mesenchymal stem cells are combined with metformin to restore the VEGF signaling pathway, MAPK signaling pathway, and mTOR signaling pathway in DFU patients through paracrine function, and inhibit the P53 signaling pathway to promote wound angiogenesis and healing.

[0033] A fifth object of the present invention is to provide a pharmaceutical composition for treating diabetic foot ulcers and / or lowering blood sugar.

[0034] To achieve the above object, the present invention adopts the following technical solutions:

[0035] A pharmaceutical composition for treating diabetic foot ulcers and / or lowering blood sugar, comprising human umbilical cord mesenchymal stem cells and metformin.

[0036] Preferably, the pharmaceutical composition is a combination drug; or the pharmaceutical composition is prepared in the form of a compound drug into any pharmaceutically acceptable dosage form.

[0037] The beneficial effects of the present invention are:

[0038] 1. This study established a diabetic foot ulcer rat model and, through in vivo and in vitro experiments and bioinformatics analysis, demonstrated for the first time that hUC-MSCs combined with metformin have a synergistic effect in the treatment of diabetic foot ulcers. This study provides a theoretical basis and experimental support for the development of more effective treatment options.

[0039] 2. Based on the synergistic therapeutic potential of hUC-MSCs and metformin, this paper further explores the mechanism of action of hUC-MSCs and its combined pharmacodynamic dose on wound healing and angiogenesis in the DFU rat model through in vitro and in vivo experiments, providing new ideas and experimental support for the treatment of diabetic foot ulcers.

[0040] 3. This study found that hUC-MSCs, and their combination with metformin, effectively promoted wound healing in rats with diabetic fusarium ulcers (DFUs). hUC-MSCs promoted wound closure, re-epithelialization, collagen deposition, angiogenesis, and relief of chronic inflammation in DFU rats. Metformin also exerted a synergistic effect by improving diabetic symptoms and systemic metabolic status.

[0041] 4. This study found that hUC-MSCs and their combination with metformin effectively promoted angiogenesis in DFU rat wounds. hUC-MSCs promote the dynamic regulation of angiogenesis and wound healing through paracrine secretion. Metformin, as an adjunctive therapy, further enhanced the therapeutic effect by prolonging the retention of hUC-MSCs in wounds exposed to high glucose and restoring the paracrine function of hUC-MSCs, which is impaired in high glucose environments.

[0042] 5. The present invention found that hUC-MSCs also significantly improved the survival, proliferation and migration ability of HUVEC in a high-glucose environment through a paracrine mechanism, and metformin further exerted a synergistic effect in promoting angiogenesis in vitro by promoting the viability and function of HUVEC cells in a high-glucose environment. Further studies found that hUC-MSCs combined with metformin regulate angiogenic PI3K-AKT and its downstream signaling pathways, including VEGF, MAPK, mTOR, and P53-related pathways, thereby promoting the enhancement of new blood vessel formation in vivo and in vitro. Metformin can enhance the regulatory effect of hUC-MSCs on these signaling pathways, synergistically promoting angiogenesis and wound healing. The two exert a synergistic effect. These findings provide new ideas and experimental basis for subsequent in-depth research on its mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results of hUC-MSCs cell characterization experiments are shown in Figure 2. Figure 1 -A is the morphology of hUC-MSCs at passage 5, the scale bar is 200 μm; Figure 1 -B is the result of detecting the proliferation ability of hUC-MSCs by EDU method, the scale bar in the figure is 100 μm; Figure 1 -C is a figure showing the expression of CD73 on the surface of hUC-MSCs identified by flow cytometry; Figure 1 -D is a figure showing the expression of CD90 on the surface of hUC-MSCs identified by flow cytometry; Figure 1 -E is a figure showing the expression of CD105 on the surface of hUC-MSCs identified by flow cytometry; Figure 1 -F is a figure showing the expression of CD34 on the surface of hUC-MSCs identified by flow cytometry; Figure 1 -G is a graph showing the expression of CD45 on the surface of hUC-MSCs identified by flow cytometry; Figure 1 -H is a graph showing the expression of HLA-DR on the surface of hUC-MSCs identified by flow cytometry. All experiments were repeated three times.

[0044] Figure 2 This is the result of the three-lineage transdifferentiation ability test of hUC-MSCs cells, among which, Figure 2 -A is the result of oil red O staining to detect adipogenesis induction; Figure 2 -B is the result of osteogenic induction detection by Alizarin red staining; Figure 2 -C is the result of Alcian blue staining to detect chondrogenic induction; the scale bar in the figure is 400 μm, and all experiments were repeated 3 times;

[0045] Figure 3 This is the evaluation result diagram of the changes in biological indicators of rats, among which, Figure 3 -A is the experimental scheme flow chart; Figure 3 -B is the test result of body weight after STZ injection; Figure 3 -C is the test result of fasting blood glucose value after STZ injection; Figure 3 -D is the result of intraperitoneal glucose tolerance test at 8 weeks after STZ injection; Figure 3 -E is the test result of insulin resistance index at 8 weeks after STZ injection; Figure 3 -F is the test result of daily water intake in the 8th week after STZ injection; Figure 3 -G is the result of the daily urine volume test at 8 weeks after STZ injection; all experiments were repeated 3 times; Student's t-test was used to compare the significance between the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001;

[0046] Figure 4 The figure shows the test results of the wound closure changes in DFU rats, where: Figure 4 -A is the wound images of rats at different time points; Figure 4 -B is a diagram of a rat wound tracing model; Figure 4 -C is a graph of the wound closure rate of rats; all experiments were repeated 3 times; Student's t-test was used to compare the significance of the experiments between the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001;

[0047] Figure 5 The figure shows the experimental results of wound re-epithelialization on the 19th day after treatment in rats, among which: Figure 5 -A is the result of H&E staining to evaluate the epithelialization tissue structure of rat wounds. In the figure, E is the epidermis, P is the superficial layer of the dermis, R is the reticular layer, N is normal skin tissue, H is the healed skin tissue, W is the unhealed skin tissue, new sebaceous glands (black arrows) and hair follicles (red arrows). The scale bar in the figure is 400 μm. Figure 5 -B is a graph for quantitative analysis of scar width in the center of the wound; Figure 5 -C is a graph for quantitative analysis of the number of newly formed hair follicles in the wound; Student's t-test was used to compare the significance of the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates no significance;

[0048] Figure 6 This is the result of the collagen deposition change test on the 19th day after treatment in rats, where: Figure 6 -A is the result of Masson trichrome staining to detect new collagen deposition in rat dermis. In the figure, blue represents collagen fibers, red represents muscle fibers, and the scale bar is 200 μm. Figure 6 -B is a figure showing the quantitative analysis of the average area of ​​dermal collagen; all experiments were repeated three times; Student's t-test was used to compare the significance of the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates no significance;

[0049] Figure 7 This is a diagram showing the expression levels of ColⅠ, ColⅢ, MMP2, MMP3, and MMP9 in rat wounds detected by Western blot and their quantification results; Figure 7 -A is a graph showing the expression levels of ColⅠ, ColⅢ, MMP2, MMP3, and MMP9 in rat wounds detected by Western blot;

[0050] Figure 7-B is a graph showing the quantitative analysis of the expression levels of ColⅠ, ColⅢ, and ColⅢ / Ⅰ in rat wounds; Figure 7 -C is a graph showing the quantitative analysis of the expression levels of MMP2, MMP3, and MMP9 in rat wounds; Student's t-test was used to compare the significance of the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significance;

[0051] Figure 8 The figure shows the experimental results of the changes in cells and cytokines related to wound healing on the 14th day after treatment in rats, among which: Figure 8 -A is a graph showing the expression of Ki67 in rat wounds detected by immunohistofluorescence staining, with the scale bar representing 100 μm; Figure 8 -B is Figure 8 -A quantitative analysis result diagram; Figure 8 -C is a graph showing the expression of rat wound cell marker proteins CD31, CD34, α-SMA, FSP1, K14, CD163, and CD86 detected by Western blot; Figure 8 -D is Western blot analysis of the expression of cytokines VEGFA, TGF-β, bFGF, IL-10, IL-1B, and TNF-α in rat wounds; all experiments were repeated three times; Student's t-test was used to compare the significance of the two groups, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significance;

[0052] Figure 9 The graph shows the changes in wound angiogenesis on the 7th and 14th days after treatment in rats, Figure 9 -A is a graph showing the expression of CD31 and α-SMA in the wounds of rats on the 7th day after treatment by immunohistofluorescence staining. The scale bar in the figure is 100 μm. Figure 9 -B is a graph showing the expression of CD31 and α-SMA in the wounds of rats on the 14th day after treatment by immunohistofluorescence staining. The scale bar in the figure is 100 μm. Figure 9 -C is a figure showing quantitative analysis of CD31-positive blood vessels; Figure 9 -D is a quantitative analysis of CD31 and α-SMA double-positive blood vessels; Figure 9 -E is a diagram of the quantitative analysis of the lumen structure cross-section; all experiments were repeated three times; Student's t-test was used to compare the significance of the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates no significance;

[0053] Figure 10 This is a graph showing changes in angiogenesis-related proteins in the wound of rats on the 14th day after treatment, among which: Figure 10 -A is a graph showing the expression of VEGFA in rat wounds detected by immunohistofluorescence staining on day 14, with the scale bar being 100 μm; Figure 10 -B is Figure 10 -A's quantitative results graph;

[0054] Figure 10 -C is a graph showing the expression of cytokines VEGFR2, P-VEGFR2, eNOS, MMP9, and MMP2 in the wounds of rats on day 14 detected by Western blot; all experiments were repeated three times; Student's t-test was used to compare the significance of the two groups, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significance;

[0055] Figure 11 The figure shows the experimental results of the effect of Met on the retention of hUC-MSCs in rat wounds after 3 days and 7 days. Figure 11 -A is a graph showing the expression of EdU and K14 in rat wounds detected by immunohistofluorescence staining, with the scale bar representing 200 μm; Figure 11 -B is a graph showing the half-inhibitory concentration of glucose on hUC-MSCs detected by MTT assay; Figure 11 -C is the result of MTT assay to detect the effect of different concentrations of Met on the viability of hUC-MSCs cells; Figure 11 -D is the result of MTT assay to detect the effect of Met treatment on the proliferation of hUC-MSCs in a high glucose environment; all experiments were repeated three times; Student's t-test was used to compare the significance between the two groups, * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significance;

[0056] Figure 12 The figure shows the experimental results of the effect of Met on the cell function of hUC-MSCs transplanted rat wounds on the 7th day. Figure 12 -A is a graph showing the expression of EdU and CD31 in rat wounds detected by immunohistofluorescence staining, with the scale bar representing 100 μm; Figure 12 -B is a graph showing the expression of EdU and α-SMA in rat wounds detected by immunohistofluorescence staining, with the scale bar representing 100 μm; Figure 12 -C is a Western blot analysis of the expression of VEGFR2, P-VEGFR2, eNOS, MMP9, and MMP2 in hUC-MSCs cells treated with Met in a high glucose environment; Figure 12 -D is Figure 12-C Quantification results; All experiments were repeated 3 times; Student's t-test was used to compare the significance of the two groups, * indicates p < 0.05, *** indicates p < 0.001;

[0057] Figure 13 The results of the effects of MSC-CM and its combination with Met on the proliferation of HUVEC cells are shown in Figure 2. Figure 13 -A is a graph showing the half-inhibitory concentration of glucose on HUVEC detected by MTT assay; Figure 13 -B is a graph showing the effect of different concentrations of Met on HUVEC cell viability detected by MTT assay; Figure 13 -C is a graph showing the proliferation ability of HUVEC detected by EDU method, with the scale bar representing 100 μm; Figure 13 -D is Figure 13 -C's quantitative results graph; Figure 13 -E is a graph showing the proliferation ability of HUVECs detected by plate colony formation assay, with the scale bar representing 5 mm; Figure 13 -F is Figure 13 -E quantitative result diagram; Figure 13 -G is a graph showing the expression of apoptosis-related proteins Bcl-2 and Bad in HUVEC cells detected by Western blot. All experiments were repeated three times. Student's t-test was used to compare the significance of the two groups. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significance.

[0058] Figure 14 Figure 2 is the experimental results of the effects of MSC-CM and its combination with Met on the migration ability of HUVEC cells, where: Figure 14 -A is a graph showing the scratch healing ability of HUVEC detected by scratch assay, with the scale bar representing 200 μm; Figure 14 -B is Figure 14 -A's quantitative results graph; Figure 14 -C is a figure showing the migration ability of HUVEC cells detected by Transwell assay, with the scale bar being 100 μm; Figure 14 -D is Figure 14 -C's quantitative results graph; Figure 14 -E is a graph showing the expression of HUVEC cell migration-related proteins E-cadherin, N-cadherin, Vimentin, MMP9, and MMP2 detected by Western blot. All experiments were repeated three times. Student's t-test was used to compare the significance between the two groups. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0059] Figure 15Figure 2 is the experimental results of the effects of MSC-CM and its combination with Met on the angiogenesis ability of HUVEC cells, where: Figure 15 -A is the result of angiogenesis assay to detect the angiogenesis ability of HUVEC in vitro, the scale bar in the figure is 100 μm; Figure 15 -B is a diagram for quantitative analysis of the number of nodes in the HUVEC vascular network; Figure 15 -C is a graph for quantitative analysis of the number of lumens in the HUVEC vascular network; Figure 15 -D is a graph for quantitative analysis of the total length of the HUVEC vascular network; Figure 15 -E is a Western blot analysis of the expression of HUVEC angiogenesis-related proteins VEGFA, TGF-β, VEGFR2, and p-VEGFR2; all experiments were repeated three times; Student's t-test was used to compare the significance between the two groups, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001;

[0060] Figure 16 This is the RNA-seq analysis result of HUVEC cells in a high glucose environment after MSC-CM treatment, where: Figure 16 -A is a cluster heat map summarizing the DEGs in the normal group and HG group, and HG group and MSC-CM group; Figure 16 -B is a volcano plot showing the DEGs between the normal group and the HG group. In the figure, the X-axis represents the p-value of the logarithmic transformation, the Y-axis represents the fold of DEG, the gray dots represent DEGs that are not differentially expressed, the red dots represent up-regulated DEGs, and the blue dots represent down-regulated DEGs; Figure 16 -C is the volcano plot of DEGs in the HG group and MSC-CM group; Figure 16 -D is a bar graph showing the enrichment of GO pathways of DEGs between the HG group and the MSC-CM group; Figure 16 -E is a bubble plot showing the KEGG pathway enrichment of DEGs between the HG group and the MSC-CM group;

[0061] Figure 17 Figure 1 is the PI3K-AKT signaling pathway diagram of DEGs between HG and MSC-CM groups;

[0062] Figure 18 This is a diagram of Western blot experiments to detect PI3K-AKT and its downstream signaling pathways in HUVEC cells, where: Figure 18 -A is a graph showing the expression levels of AKT, p-AKT, eNOS, p-eNOS, P53, and GAPDH in HUVEC cells detected by Western blot assay;

[0063] Figure 18-B is a graph showing the expression levels of mTOR, p-mTOR, ERK1 / 2, p-ERK / 2, and α-Tubulin in HUVEC cells detected by Western blot assay; all experiments were performed in triplicate;

[0064] Figure 19 This is the result of immunohistochemical experiment to detect PI3K-AKT and its downstream signaling pathway in rat wounds. The scale bar in the figure is 50μm.

[0065] Figure 20 for Figure 19 Quantification results are shown. All experiments were repeated three times. Student's t-test was used to compare the significance between two groups. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates no significance. DETAILED DESCRIPTION

[0066] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0067] In the examples of the present invention, unless otherwise specified, the reagents, materials, instruments, cells (such as HUVEC cells), etc. involved in the experiments can be purchased through conventional commercial channels.

[0068] This study, centered around hUC-MSCs and the hypoglycemic drug Met, uses a diabetic foot ulcer (DFU) rat model and HUVEC cells in a high-glucose environment as research subjects. Through in vivo and in vitro experiments, we explored the effects and mechanisms of both, and their combined treatment, on wound healing and angiogenesis in diabetic foot ulcers. First, hUC-MSCs were isolated, prepared, and identified from umbilical cord tissue, strictly adhering to the principles of ethical review and approval by the ethics committee and informed consent of the donors. Second, based on the successful establishment of a rat model of diabetic foot ulcers, in vivo experiments preliminarily evaluated several key aspects of the DFU rat model, including wound closure, re-epithelialization, matrix remodeling, and proliferation of vascular and inflammatory-related cells. Furthermore, we further evaluated the dynamic regulation of angiogenesis in DFU rats, as well as the retention, engraftment, and function of hUC-MSCs in the subcutaneous vascular structure of the wound. Finally, in vitro experiments further explored the effects of hUC-MSC culture supernatant (MSC-CM), Met, and their combined treatment on the survival, proliferation, migration, and angiogenesis of HUVEC cells in a high-glucose environment. Finally, RNA-seq, immunoblotting, immunohistochemistry and other experimental techniques were used to further reveal the signaling pathway mechanism of hUC-MSCs and their combined treatment in promoting angiogenesis in vitro and in vivo at the molecular level. The specific experimental results are as follows:

[0069] (1) The cell morphology, phenotype and multilineage differentiation potential of hUC-MSCs meet the definition criteria.

[0070] hUC-MSCs cells were isolated and prepared by the tissue block adhesion method, and they were able to grow adherently and were in a typical long spindle shape. EdU labeling detection showed that they had good in vitro proliferation ability. Flow cytometry identification of hUC-MSCs cell phenotype showed that their surface specific markers CD73, CD90, and CD105 were positive, and hematopoietic markers and immune rejection-related markers CD34, CD45 and HLA-DR were negative, which met the mesenchymal stem cell identification standards of the International Society for Cell Therapy (ISCT). In vitro induction experiments confirmed that they can differentiate into adipogenic, osteogenic and chondrogenic cells, and have multi-lineage transdifferentiation capabilities. In summary, the present invention successfully prepared hUC-MSCs cells and performed EdU labeling, which is consistent with the state, characteristics and functions of mesenchymal stem cells, laying the foundation for subsequent transplantation and in-depth research.

[0071] (2) hUC-MSCs and combined treatment promoted wound closure, re-epithelialization and matrix remodeling, and proliferation of vascular and inflammation-related cells in DFU rats. Met could exert a combined therapeutic effect by improving the systemic metabolic status.

[0072] First, type 2 diabetes was induced by a combination of high-fat diet and STZ injection. Subsequently, foot ulcers were created to establish a DFU rat model. The reliability of the model was verified by physiological indicators and histology. This model exhibited highly similar pathological conditions to diabetic foot ulcers in terms of weight loss, hyperglycemia, impaired glucose metabolism, insulin resistance, polydipsia and polyuria, as well as delayed wound healing, unstable epithelialization, impaired angiogenesis, and a chronic inflammatory microenvironment. This indicates that the model was successfully constructed and provides a reliable animal experimental basis for in-depth research on subsequent therapeutic interventions. Secondly, it was found that subcutaneous muscle transplantation of hUC-MSCs (approximately 2×10 6 cells / kg) and its combination with Met in the wound healing of diabetic foot ulcers. DFU rats injected with hUC-MSCs cells did not develop skin tumors during the experiment, and there were no obvious adverse reactions or transplant rejection reactions. The effects of hUC-MSCs and its combination with Met on re-epithelialization and tissue remodeling in the wound healing process of DFU rats were evaluated by wound images and immunohistology. Compared with the control group, the MSC treatment group and the combination treatment group showed significant advantages in wound area reduction, wound closure rate, accelerated epithelialization, regeneration of skin appendages (such as hair follicles), dermal collagen deposition, anti-scar formation, and regulation of matrix metalloproteinases (MMPs) expression. This shows that hUC-MSCs transplantation and combination treatment can effectively improve the tissue repair and regeneration ability of diabetic foot ulcer skin and skin function, and significantly improve the scar-free healing quality of diabetic foot ulcer wounds. In addition, immunohistofluorescence and immunoblotting were used to deeply explore the proliferation activities of key cells and the expression of related cytokines in the proliferation stage of wound healing. Compared with the control group, cell proliferation was active in the MSC-treated group and the combined-treatment group, and the expression levels of markers of vascular endothelial cells, smooth muscle cells, fibroblasts, keratinocytes and M2 anti-inflammatory macrophages were higher, while the expression level of M1 pro-inflammatory macrophage markers was lower. The expression levels of related angiogenic factors and cytokines also showed a trend conducive to wound healing. This further indicates that subcutaneous transplantation of hUC-MSCs and combined treatment contribute to angiogenesis, re-epithelialization, macrophage polarization and relief of chronic inflammation during wound healing. These effects were further enhanced in the wounds of DFU rats treated with combined treatment, and their diabetic conditions such as hyperglycemia, impaired glucose metabolism, and insulin resistance were significantly alleviated, indirectly indicating that metformin can exert a synergistic effect by improving the systemic metabolic state.

[0073] (3) hUC-MSCs promote the dynamic regulation of angiogenesis and healing of DFU rat wounds through paracrine secretion, and Met can exert a combined therapeutic effect by improving the retention and paracrine function of hUC-MSCs in the wound.

[0074] Neovascularization plays a key role in wound healing. Immunohistofluorescence staining and immunoblotting revealed that vascular density, number, and cross-sections in DFU rats were significantly lower than those in the normal control group on days 7 and 14 after treatment, indicating severe impairment in the establishment and maintenance of a functional vasculature. High expression levels of MMP2 and MMP9 on day 19 suggest that angiogenic cells may migrate to form new vessels. The hUC-MSCs-treated and combined treatment groups showed a significant increase in the number of angiogenic cells and vessels on day 7, and continued expansion of the vascular structure on day 14. The expression levels of VEGFA, VEGFR2, p-VEGFR2, eNOS, MMP2, and MMP9 were significantly higher than those in the control group. However, MMP2 and MMP9 expression levels were significantly decreased on day 19, indicating a gradual weakening of angiogenesis. Thus, hUC-MSCs treatment not only promotes the proliferative phase of angiogenesis but also accelerates the maturation phase, effectively ameliorating vascular dysfunction in DFU rats. Met promotes this effect through combined treatment. Secondly, the retention of hUC-MSCs in DFU rat wounds was investigated by fluorescence co-staining experiments with K14 and EdU. On the third day after transplantation, hUC-MSCs were primarily distributed in the superficial dermis and rapidly decreased over time. On the seventh day, some cells migrated and implanted into the deep reticular layer of the wound site. Combined treatment with Met prolonged the retention of cells and enhanced their implantation in the wound site. In vitro MTT experiments further revealed the promoting effect of Met on hUC-MSC cell viability in a high-glucose environment, indicating that Met could significantly restore the inhibitory effect of high-glucose on hUC-MSC cell proliferation. Finally, the engraftment and mechanism of action of hUC-MSCs in DFU rat wounds were investigated by fluorescence co-staining experiments with CD31, α-SMA, and EdU. hUC-MSCs migrated into the deep vascular structures of the wound dermis and participated in angiogenesis through paracrine effects. Immunoblotting confirmed that the hUC-MSCs isolated and cultured in this study possess robust paracrine activity, secreting multiple angiogenesis-related factors, including VEGFA, TGF-β, bFGF, and IL-10, to promote angiogenesis and wound healing in rats with DFU. High glucose significantly inhibited the paracrine function of hUC-MSCs. Met could be used as a co-therapy to restore the paracrine function of hUC-MSCs impaired by high glucose, further enhancing the therapeutic effect. These findings provide new insights and experimental basis for further investigation of the underlying mechanisms.

[0075] (4) hUC-MSCs promote the proliferation, migration and angiogenesis of vascular endothelial cells through paracrine secretion, and Met can exert a combined therapeutic effect by promoting the viability and function of vascular endothelial cells.

[0076] Angiogenesis is a key component in wound healing research for diabetic foot ulcers. However, hyperglycemia impairs endothelial cell viability and function, hindering angiogenesis. The serum-free culture supernatant of hUC-MSCs (MSC-CM) contains multiple cell-secreted factors, including previously validated VEGFA, TGF-β, bFGF, and IL-10. First, MTT assays, EdU detection, plate cloning assays, and immunoblotting revealed that MSC-CM and combined treatment groups significantly restored HUVEC proliferation, as demonstrated by increases in the percentage of EdU-positive cells and the number of cell clones, as well as significant upregulation of the anti-apoptotic protein Bcl-2 and downregulation of the pro-apoptotic protein Bad, alleviating the damage induced by high glucose. Second, Transwell and wound wound assays revealed that MSC-CM and combined treatment groups significantly restored HUVEC migration, as demonstrated by increases in the wound healing rate and the number of migrating cells in the Transwell, significantly promoting the expression of migration-related proteins and enhancing HUVEC motility and migration. Finally, through Matrigel tube formation assays and immunoblotting, it was found that the MSC-CM treatment group and the combined treatment group significantly restored the angiogenic capacity of HUVEC, as demonstrated by a significant increase in the number of vascular nodes, lumen, and total length, upregulation of VEGFA, TGF-β, and p-VEGFR2 expression, and activation of the VEGF / VEGFR signaling pathway, thereby promoting HUVEC angiogenesis in vitro. Therefore, hUC-MSCs significantly improve the survival, proliferation, and migration of HUVEC in a high glucose environment through a paracrine mechanism, thereby promoting in vitro angiogenesis, while Met exerts a combined therapeutic effect by promoting the viability and function of HUVEC cells in a high glucose environment.

[0077] (5) hUC-MSCs promote PI3K-AKT and its downstream signaling pathways of angiogenesis in vitro and in vivo through paracrine secretion, and Met exerts a combined therapeutic effect.

[0078] First, RNA-seq results were used to analyze the DEGs between the high glucose (HG) and MSC-CM-treated HUVEC cells, and pathway enrichment analysis revealed the primary role of the PI3K-AKT signaling pathway. Secondly, immunoblotting experiments validated the RNA-seq analysis results, revealing that the expression levels of p-AKT, p-eNOS, p-mTOR, and p-ERK1 / 2 in HUVEC cells were significantly decreased under high glucose conditions, while the expression level of P53 was significantly increased. MSC-CM treatment significantly restored the activity of the above signaling pathways in HUVEC cells under high glucose conditions, promoting functional recovery of HUVEC cells, including cell survival, proliferation, migration, and angiogenesis. Furthermore, co-treatment with Met further enhanced the regulatory effects of MSC-CM on these signaling pathways. Finally, immunohistochemistry experiments further validated the above in vitro experimental results in a DFU rat model. In the wounds of DFU rats, the hUC-MSCs and combined treatment groups showed significantly higher expression of p-AKT, p-eNOS, p-mTOR, and p-ERK1 / 2 than in the control and Met groups, while p53 expression was significantly reduced. In vivo experiments demonstrated that hUC-MSCs significantly restored the activity of PI3K / AKT downstream signaling pathways, including VEGF, MAPK, and mTOR, in the wounds of DFU rats through a paracrine mechanism and inhibited the P53 apoptosis pathway. These findings are consistent with previously reported experimental results, demonstrating that Met exerts a combined therapeutic effect, further enhancing the regulatory effects of hUC-MSCs on these signaling pathways and promoting angiogenesis and wound healing.

[0079] Example 1. Preparation, characterization and identification of human umbilical cord mesenchymal stem cells

[0080] 1. Experimental Materials

[0081] (1) Human umbilical cord mesenchymal stem cells

[0082] Human umbilical cord mesenchymal stem cells are prepared from umbilical cord tissue samples obtained from the obstetrics and gynecology department and are derived from the portion of the umbilical cord that is severed after natural childbirth or cesarean section. Pregnant women screened are 35 years of age or younger, with normal physical examinations, no history of obvious infectious diseases or family genetic diseases, and no history of gestational diabetes or hypertension. Sample collection is carried out in compliance with national laws and regulations, and has been reviewed and approved by the Medical Ethics Committee, and the donor's "Informed Consent" has been obtained. Clean bench

[0083] (2) Experimental solution formula

[0084] hUC-MSCs cell culture medium formula: MEM-Alpha medium 45ml, fetal bovine serum 5ml and P / S 500ul.

[0085] Cell freezing solution formula: DMSO 200ul, FBS 1800ul.

[0086] 1× phosphate buffered saline (PBS) recipe: 10g 10x PBS, 1L H2O.

[0087] Formula of 4% paraformaldehyde solution: PFA powder 4g, 1×PBS 100ml.

[0088] Formula of 0.3% TritonX-100 solution (0.3% Triton solution): TritonX-1000 0.3 ml, 1×PBS 100 ml.

[0089] 2. Experimental Methods

[0090] 1. Isolation and culture of hUC-MSCs

[0091] (1) Umbilical cord tissue pretreatment

[0092] In a sterile cell operation room, the healthy umbilical cord was first thoroughly rinsed with sterilized 1×PBS solution to remove residual blood, and then both ends of the umbilical cord tissue were clamped and soaked in 75% alcohol for about 5 minutes. Then, the cord was rinsed twice with 1×PBS and cut into small segments of about 1-3 cm. The umbilical cord was cut along the umbilical vein, the umbilical vein and umbilical artery were removed, and the cord was rinsed twice with 1×PBS.

[0093] (2) Tissue patch

[0094] In a clean bench, the umbilical cord tissue was cut into approximately 1 mm 3 For large and small tissue pieces, first suspend them in complete culture medium, then use tissue forceps to place the small umbilical cord piece on the bottom of the culture bottle and let it stand for 30 to 60 minutes.

[0095] (3) Primary culture

[0096] Gently add complete culture medium to the culture flask until the tissue is submerged and gently place it in the incubator. On the 5th to 6th day of culture, when spindle cells appear around the tissue block, perform a half-dose culture. First, aspirate half of the culture medium and then add an equal amount of culture medium that has returned to room temperature, taking care to avoid touching the tissue block that is attached to the wall. Then, replace the medium with a full volume every 2-3 days and observe cell growth. When the cells in the local area around the tissue block are approximately 60%-80% confluent, proceed to cell passage.

[0097] (4) Subculture

[0098] ① Trypsin digestion: Subculture the cells when they are about 80% confluent. Wash them 1-2 times with sterile 1× PBS, then add trypsin digestion solution and digest for about 2-3 minutes until the cells become round and begin to fall off the culture flask. Add an equal amount of complete culture medium to terminate the digestion.

[0099] ② Wash with 1× PBS: Collect the digested cell suspension in a centrifuge tube and centrifuge at 150×g for 5 minutes. Discard the supernatant, resuspend the cells in 1× PBS, and centrifuge again.

[0100] ③ Cell Seeding: Resuspend the cells in complete culture medium and seed them proportionally into new culture dishes. Continue culturing in a cell culture incubator and observe cell growth. Change the medium every two days. When the confluency reaches approximately 80%, subculture again. Cells from passages 3 to 7 are used for experimental research.

[0101] (5) Cell cryopreservation

[0102] When the cell fusion rate reaches 80%-90%, the cells are digested, washed and centrifuged, and then the cell freezing solution is added and mixed. The cells are transferred to cryopreservation tubes and placed in a freezing box. For short-term storage at -80°C, for long-term storage, after 48 hours, the cryopreservation tubes are transferred from -80°C to a liquid nitrogen tank.

[0103] 2. EdU cell proliferation assay

[0104] (1) hUC-MSCs were digested and washed and then seeded into 24-well plates, with 2×10 cells per well. 4 cells, repeated 3 times, and then placed them in a cell culture incubator to adhere to the wall.

[0105] (2) After 24-48 hours of adherent growth, EdU (10 mM) was diluted 1:1000 with cell culture medium and preheated before being transferred to a 24-well plate for cell labeling. The cells were incubated in a cell culture incubator for another 2 hours and then removed.

[0106] (3) Add 4% PFA fixative and remove the solution after 15 minutes at room temperature. Wash the cells with 1× PBS and then remove the solution.

[0107] (4) Add 0.3% Triton X-100, remove it after 15 minutes, and then wash the cells with 1× PBS and remove it.

[0108] (5) Prepare the Click reaction solution according to Table 1. Prepare it immediately before use. Add 0.5 ml of the reaction solution and shake gently to evenly cover the well plate. Incubate at room temperature in the dark for 30 minutes, then add 1× PBS to wash and remove.

[0109] Table 1. Click reaction solution

[0110] Components Dosage ClickReactionBuffer 1.72ml <![CDATA[CuSO4]]> 80ul Azide488 4ul ClickAdditiveSolution 200ul Total volume 2ml

[0111] (6) Add 1 ml of 1× Hoechst to each well, incubate at room temperature in the dark for 10 minutes, and then wash the cells three times with 1× PBS, each time for 3-5 minutes.

[0112] (8) Fluorescence detection was performed and the cell proliferation rate was calculated as EdU-positive cells (green fluorescence) / Hoechst-positive total cells (blue fluorescence)*100%.

[0113] 3. Flow cytometry

[0114] (1) After digestion and washing, hUC-MSCs were divided into blank tubes, single staining tubes, IgG isotype control tubes, and flow cytometry tubes. The cells were centrifuged at 4°C, 150×g, and 5 min, and the supernatant was removed.

[0115] (2) Add 100 μl of 1× PBS to prepare a single-cell suspension, then add appropriate amounts of CD73, CD105, CD90, CD45, CD34, and HLA-DR antibody staining solutions to resuspend the cells and label them, and incubate in the dark for 30 min.

[0116] (3) After the incubation, 400 μg of each of the above cell suspensions was added. The cells were diluted with 1×PBS and filtered through a cell sieve before detection by flow cytometry. The experimental results were analyzed using FlowJ software.

[0117] 4. Adipogenic Differentiation and Oil Red O Staining

[0118] (1) Prepare adipogenic differentiation medium solutions A and B according to Tables 2 and 3. Mix thoroughly and store at 4°C. Preheat to 37°C before use. If granular precipitates form in additive A-II, re-dissolve by repeatedly pipetting after a brief 37°C water bath.

[0119] Table 2. Adipogenic differentiation medium A

[0120] Components Dosage Cell basal culture medium 177ml Fetal bovine serum 20ml Adipogenic differentiation supplement AI 3ml Adipogenic Differentiation Supplement A-II 200ul

[0121] Table 3. Adipogenic differentiation medium B

[0122] Components Dosage Cell basal culture medium 90ml Fetal bovine serum 10ul Adipogenic Differentiation Supplement B 200ml

[0123] (2) Add 1 ml of 0.1% gelatin to a 12-well plate and cover it evenly. Place it on a clean bench for 30 to 60 minutes, then remove the solution and air dry the plate.

[0124] (3) After counting the hUC-MSCs cells, they were seeded into the well plate to a volume of 1×10 4 cells / cm, supplement with 1 ml of complete cell culture medium and place in the incubator.

[0125] (4) When the cell confluency is approximately 100%, carefully remove the complete medium from the wells and add 1 ml of adipogenic differentiation medium A. Induce for 3 days. Then, remove medium A from the wells and add 1 ml of adipogenic differentiation medium B. After inducing for 1 day, remove medium B and replace with medium A. Continue alternating culture in a pattern of "3 days in medium A, 1 day in medium B."

[0126] (5) Observe the cell status until sufficient lipid droplets of appropriate size appear. Remove the differentiation medium from the wells by aspirating, add 1× PBS for washing, and then remove.

[0127] (6) Add 4% PFA solution, fix for 10 minutes, then remove it, and then wash it with 1× PBS and remove it.

[0128] (7) According to the instructions of the Oil Red O staining kit, add the Oil Red O staining working solution. After 10-20 minutes, add an appropriate amount of staining washing solution, let it stand for 30 seconds, and then remove it by aspiration.

[0129] (8) After washing twice with 1× PBS, observe and photograph under a microscope.

[0130] 5. Osteogenic differentiation and Alizarin Red Staining

[0131] (1) Prepare osteoinduction differentiation medium according to Table 4, mix thoroughly and store at 4°C.

[0132] Table 4. Osteogenic differentiation medium

[0133] Components Dosage Cell basal culture medium 177ml Fetal bovine serum 20ml Osteogenic differentiation supplements 3ml

[0134] (2) Coat a 12-well plate with 0.1% gelatin and seed hUC-MSCs cells to 1×10 4 cells / cm.

[0135] (3) When the cell confluence reaches 70%, carefully remove the complete medium from the well and add 1 ml of osteogenic differentiation medium. Change the medium every 3 days. When obvious calcium nodules appear, change the medium with half the volume every 2 days.

[0136] (4) After 2-4 weeks, observe the cell morphology and growth, wash with 1×PBS, add 4% PFA solution, fix for 30 minutes, and then wash again with 1×PBS.

[0137] (5) Add Alizarin Red staining solution and wash with 1× PBS after about 10 minutes.

[0138] (6) Add an appropriate amount of 1× PBS, observe under a microscope and take pictures.

[0139] 6. Chondrogenic Differentiation and Alcian Blue Staining

[0140] (1) Prepare the induction medium premix according to Table 5 and store at 4°C. Osteogenic differentiation supplement II should be aliquoted and stored at -20°C. Before use, add it to the premix at a ratio of 100:1 to prepare the induction differentiation medium.

[0141] Table 5. Chondrogenic differentiation medium premix

[0142] Components Dosage Cell basal culture medium 97ml Osteogenic Differentiation Supplement I 2ml

[0143] (2) Inoculate approximately 3 to 4 × 10 cells into a 15 ml centrifuge tube. 5 Cells were centrifuged at 150 × g for 5 min.

[0144] (3) Add 0.5 ml of chondrogenic differentiation premix, resuspend and wash, then centrifuge at 150 × g for 5 min. Repeat the washing step once to obtain a cell pellet.

[0145] (4) Add 0.5 ml of complete differentiation medium to resuspend the cells and centrifuge at 150 × g for 5 min. Gently remove the centrifuge tube and loosen the cap to facilitate gas exchange. Place the tube upright in a cell culture incubator for 24 h until the cells aggregate.

[0146] (5) After 24-48 hours, gently tap the bottom of the centrifuge tube to detach the chondrocytes from the bottom of the tube and suspend them in the liquid. Replace the induction medium every 2-3 days, gently tap the centrifuge tube, loosen the centrifuge tube cap and continue to place it in the incubator.

[0147] (6) Continue induction until cartilage balls with a diameter of approximately 1.5 to 2 mm are formed, which are then washed with 1× PBS and removed.

[0148] (7) Add 4% PFA solution and dehydrate for 30-60 minutes to make the sample transparent. The process is as follows: 50% alcohol, 30 minutes; 70% alcohol, 30 minutes; 80% alcohol, 30 minutes; 95% alcohol, 30 minutes; anhydrous ethanol, 30 minutes; 1 / 2 xylene, 2 hours; xylene, 1.5 hours; xylene, 1 hour. Note that 1 / 2 xylene is prepared by mixing xylene and anhydrous ethanol in a volume ratio of 1:1.

[0149] (8) Soak the cartilage ball in a paraffin embedding machine at 55°C for about 2 hours, then embed it. After cooling and forming, remove the wax block.

[0150] (9) Place the wax block in a microtome and slice it continuously, each slice being 3 μm thick. Then spread the slices onto glass slides, dry them in a 55°C oven, and dewax them according to the following process: xylene, 15 min; xylene, 10 min; 1 / 2 xylene, 10 min; anhydrous ethanol, 10 min; 95% alcohol, 10 min; 80% alcohol, 10 min; 70% alcohol, 10 min; 50% alcohol, 10 min. Allow to air dry.

[0151] (10) Add a drop of alexian blue dye to the slice, rinse with water after 1 hour, let it dry, and observe under a microscope.

[0152] 7. Data statistical analysis

[0153] All experiments were performed in triplicate, and all data were analyzed using GraphPad Prism (version 8.0) software. The data between the two groups were compared using a two-tailed student's t-test, and P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001).

[0154] 3. Experimental Results and Analysis

[0155] (1) hUC-MSCs cell characterization and flow cytometry phenotyping

[0156] After primary culture and multiple passages, hUC-MSCs were purified to obtain high-purity cells that adhered to the wall. Observation under an inverted microscope revealed that all cells were slender, spindle-shaped, and fibroblast-like, with no cell debris or cytoplasmic vacuolation. Figure 1 -A. The cells can be stably passaged and grow in a vortex-like pattern. EdU labeling and cell proliferation capacity testing were performed on the fifth generation hUC-MSCs cells. The results are as follows Figure 1 -B At any time, the proportion of EdU labeling (green) in hUC-MSCs cells (blue) was high, about 90%, and the in vitro proliferation ability was good. In addition, the phenotype of hUC-MSCs cells was identified by flow cytometry, and the results were as follows Figure 1 -C~ Figure 1 -H, the red peak represents cells stained positive with the corresponding antibodies, while the blue peak represents the isotype control. The average percentage of positive cells shows that CD73, CD90, and CD105 are highly expressed, with positive rates of 99.9%, 99.9%, and 99.8%, respectively; CD34, CD45, and HLA-DR are all lacking in expression, with positive rates of 0.67%, 0.19%, and 0.75%, respectively, which meets the phenotypic identification criteria of hUC-MSCs cells.

[0157] (2) Tri-lineage transdifferentiation ability of hUC-MSCs

[0158] Another important indicator for identifying hUC-MSCs cells is the multi-lineage transdifferentiation potential of cells, namely the ability to differentiate into adipocytes, osteoblasts, and chondrocytes. These three cells can form fat, calcium deposits, and acidic matrix, respectively, and are stained with Oil Red-O, Alizarin Red, and Alcian Blue, respectively. The fifth generation hUC-MSCs cells were induced to adipogenesis for 15 days and stained with Oil Red-O. The results are as follows: Figure 2As shown in -A, most cells showed translucent lipid droplets of varying sizes after induction, and the obvious lipid droplets appeared bright red. This generation of cells was subjected to osteogenic induction for 20 days and stained with Alizarin red. The results are shown in Figure 2 As shown in Figure 2B, most cells developed obvious red nodules and calcium deposits after induction. The cells of this generation were cultured in chondrogenic induction medium and formed compact balls on the 22nd day of culture. The fixed sections were then stained with Alcian blue. Figure 2 As shown in Figure 3-C, most areas clearly showed blue cartilage matrix. These results indicate that hUC-MSCs have the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages.

[0159] Example 2. Effects of hUC-MSCs and their combination with Met on wound healing in DFU rats

[0160] 1. Experimental Materials

[0161] (1) Cells and Animals

[0162] Human umbilical cord mesenchymal stem cells, see Example 1.

[0163] Sprague-Dawley rats: 50 male Sprague-Dawley rats, 6-8 weeks old, SPF grade, purchased from Chongqing Enbi Biotechnology Co., Ltd. The housing environment provided appropriate ventilation, lighting, and temperature. This animal experiment was approved by the Animal Ethics Committee.

[0164] (2) Experimental solution formula

[0165] 1) 35 mg / ml STZ injection solution

[0166] ① Sodium citrate buffer: Weigh 1.05 g of anhydrous citric acid and add it to 50 ml of ultrapure water, mix well to prepare a 100 mM citric acid solution (Solution A); weigh 1.47 g of sodium citrate and add it to 50 ml of ultrapure water, mix well to prepare a 100 mM sodium citrate solution (Solution B). Before use, mix the citric acid solution and sodium citrate solution in a ratio of 1:1.32 to prepare sodium citrate buffer and adjust the pH to approximately 4.0.

[0167] ② Weigh 140 mg of STZ powder and add it to 4 ml of sodium citrate buffer to prepare 35 mg / ml STZ injection. Keep it in a dark and low temperature environment and use it within 15-30 minutes.

[0168] 2) Formula of 50mg / ml metformin gavage solution: 0.5g metformin hydrochloride tablets, 10ml ultrapure water.

[0169] 3) 40% glucose solution formula: 40g glucose, dilute to 100ml with ultrapure water.

[0170] 2. Experimental Methods

[0171] 1. Construction of diabetic foot ulcer rat model

[0172] (1) Adaptive feeding

[0173] The rats were placed in an environment with a temperature of (22±2)°C, a relative humidity of 50%-60%, and 12 hours of light / 12 hours of darkness for 1 week, with free access to food and water.

[0174] (2) Induction of type 2 diabetes

[0175] ① High-fat diet feeding: Six rats were randomly selected as the normal group and fed a regular diet. The remaining rats were selected as the model group. After 4-5 weeks of high-fat diet feeding, body weight and fasting blood glucose levels were measured. When the blood glucose level of the model group rats reached approximately 7mM, STZ was injected to induce hyperglycemia.

[0176] ② Multiple low-dose STZ injections: First, fast the rats for approximately 12 hours. After weighing the rats, accurately calculate the volume of 35 mg / ml solution required for an intraperitoneal injection of 35 mg / kg STZ per rat. Prepare the 35 mg / ml STZ injection solution immediately and administer STZ once daily for three consecutive days.

[0177] ③ Fasting blood glucose testing: Three days after the last STZ injection, rats were fasted but not watered for approximately 12 hours. Fasting blood glucose was measured at the tip of the tail using a blood glucose meter. If a single fasting blood glucose level ≥16.7 mmol / L or a weekly fasting blood glucose level ≥11.1 mmol / L was considered type 2 diabetes and the rats were included in subsequent experiments. High-fat feeding was continued for 1-2 weeks to stabilize blood glucose levels, with bedding changed daily in the morning and evening.

[0178] (3) Foot trauma surgery-induced ulcers

[0179] ① Anesthesia: First, normal rats and type 2 diabetes model rats were fasted but not watered for about 12 hours, their body weights were measured, and the volume of 10% chloral hydrate solution required for an intraperitoneal injection of 300 mg / kg chloral hydrate was accurately calculated for each rat. Each time the rat was intraperitoneally injected with anesthetic, its muscles were relaxed and sluggish.

[0180] ② Foot trauma: The rats were fixed on the operating table. After the plantar skin and surgical instruments were disinfected, a 6-mm diameter puncture biopsy instrument and surgical scissors were used to create a full-thickness skin wound of approximately 6 mm in diameter on the dorsum of the rat's foot, deep into the fascia layer, to simulate the wound of diabetic foot ulcer. After the animals regained consciousness, they were fed routinely and had free access to water and food.

[0181] ③ Postoperative care: After surgery, the rats were placed back in individual cages and given normal diet and drinking water after they woke up and were able to move normally. The rats' mental state, drinking water, weight changes, and wound conditions were continuously observed.

[0182] 2. Model grouping and treatment intervention

[0183] (1) hUC-MSCs labeling and single cell suspension preparation: Before treatment, hUC-MSCs were labeled with EdU when the confluence was about 70%-80%. EdU (10mM) was diluted 1:1000 with cell culture medium and preheated and transferred to the culture dish. The cells were incubated in the cell culture incubator for 10-12 hours. After incubation, the cells were digested, washed and centrifuged, and single cell suspension was prepared by adding pre-cooled 1×PBS. The cell concentration was adjusted to 4×10 6 / ml.

[0184] (2) Rats were grouped and weighed: On the second day after the rat foot trauma surgery, the rats were randomly divided into the following five groups according to the modeling and treatment methods: normal group, model group, metformin (Met) treatment group, stem cell (MSC) treatment group, and stem cell and metformin combined treatment group, with 6 SD rats in each group. First, each rat was weighed, and the metformin hydrochloride sustained-release solution (50 mg / ml, Table 4.4) and hUC-MSC cell suspension (4×10 6 cells / ml) to ensure that the rat Met dose was 250 mg / kg and the hUC-MSCs dose was approximately 2×10 6 cells / kg.

[0185] (3) Therapeutic intervention: The rats were first anesthetized locally on their lower limbs and then given the following treatments: the rats in the metformin treatment group were gavaged with drugs daily for 19 days; the rats in the stem cell (MSC) treatment group were injected with stem cell suspension in the subcutaneous muscles around their wounds, with approximately 6 points around each wound, and the total volume of cell suspension injected in each rat was approximately 200ul-250ul; the combined treatment group was injected with stem cells using the same method and continued to be gavaged with metformin daily, while the normal group and the model group were injected with 1×PBS using the same method.

[0186] (4) Observation and sample collection: After the treatment intervention, the rats' physiological status, including body weight, fasting blood glucose, drinking water and urine pad weight, as well as wound healing, were continuously observed and tested. After stem cell transplantation, the rats were anesthetized on the 3rd, 7th, 14th, and 19th days after treatment, and skin and blood samples from the foot wound were collected for subsequent testing.

[0187] 3. Homeostasis model assessment of insulin resistance index (HOMA-IR)

[0188] (1) Fasting blood glucose test

[0189] The rats were fasted for 12 h before and after treatment, and the fasting blood glucose levels (mmol / L) were measured by tail tip with a blood glucose meter.

[0190] (2) Fasting insulin test

[0191] ① Tail tip blood collection: Before and after treatment, rats were fasted for 12 hours. The rats were then immobilized and their tails gently massaged to promote vasodilation. The tail tip was disinfected and cut off approximately 2-3 mm to collect an appropriate amount of blood.

[0192] ②Serum separation: Let the blood stand at room temperature for 30-60 minutes. After it is completely coagulated, centrifuge it at 2000-4000 rpm for 15 minutes. Pipette the upper serum into a centrifuge tube for later use.

[0193] ③ Insulin detection: Detect insulin according to the kit instructions. After the color reaction is terminated, measure the absorbance value of each well on a microplate reader and calculate the serum insulin value (mU / L) according to the standard curve.

[0194] (3) Calculation of HOMA-IR

[0195] The calculation formula for HOMA-IR is (fasting blood glucose mmol / L x fasting insulin mU / L) / 22.5. A HOMA-IR value greater than 2.5 indicates insulin resistance, and an elevated value indicates a significantly increased risk of type 2 diabetes.

[0196] 4. Intraperitoneal glucose tolerance test (IPGTT)

[0197] (1) Calculation of glucose dosage: Before and after treatment, rats in each group were fasted for 12 h but not water, and then weighed. Based on the glucose dosage of 2 g / kg, the volume of 40% glucose solution required for intraperitoneal injection of each rat was calculated.

[0198] (2) Timed blood glucose testing: Fasting blood glucose of each group of rats was first measured using a blood glucose meter, and the blood glucose value at 0 min was recorded. Subsequently, each rat was intraperitoneally injected with 40% glucose solution according to the predetermined calculated dose. Blood glucose was then measured and recorded at 15 min, 30 min, 60 min, and 120 min. The blood collection site was immediately disinfected after the test.

[0199] (3) Draw a blood glucose-time curve and analyze the glucose tolerance of rats.

[0200] 5. Wound closure measurement

[0201] On days 3, 7, 14, and 19 after treatment, images of the paw ulcer wounds of each rat group were taken with a ruler placed over the images. Wound area was then measured using the ruler on the images. Wound size was defined as the area that did not re-epithelialize and normalized by the initial wound area. Wound closure rate at each time point = [(initial wound area – final wound area) / initial wound area] × 100%.

[0202] 6. Preparation of tissue paraffin sections and dewaxing and hydration

[0203] (1) After washing the tissue with 1× PBS, add 4% PFA solution for fixation for 24-48 hours. Dehydrate and transparentize the tissue according to the following steps: 75% alcohol, 2 hours; 85% alcohol, 2 hours; 95% alcohol, 2 hours; anhydrous ethanol, 1 hour; anhydrous ethanol, 1 hour; 1 / 2 xylene, 2 hours; xylene, 1 hour; xylene, 1 hour. 1 / 2 xylene is prepared by mixing xylene and anhydrous ethanol in a volume ratio of 1:1.

[0204] (2) Soak the tissue sample in a paraffin embedding machine at 65°C for about 12 hours, place it in a mold for embedding, let it cool and form, then remove and trim the wax block.

[0205] (3) Place the wax block in a microtome and cut it into thin slices continuously, each slice is 3-5 μm thick, and then spread it on a slide to make slices. Subsequent experiments can be carried out or stored at 4°C.

[0206] (4) After drying the sections in a 65°C oven for 4-6 hours, dewax them according to the following steps: xylene, 5 minutes; xylene, 5 minutes; 1 / 2 xylene, 5 minutes; anhydrous ethanol, 3 minutes; anhydrous ethanol, 3 minutes; 95% alcohol, 3 minutes; 85% alcohol, 3 minutes; 75% alcohol, 3 minutes.

[0207] 7. Hematoxylin-eosin staining (HE)

[0208] (1) Hematoxylin staining: Place the hydrated skin tissue sections into a hematoxylin staining solution jar and stain for 5 minutes. Then wash the sections with tap water. The cell nuclei in the sections will be stained blue.

[0209] (2) Differentiation and blueing: First, place the slices in 1% hydrochloric acid for about 10 seconds to differentiate, then soak them in dilute ammonia water for tens of seconds to make them blue, and then wash the slices with tap water.

[0210] (3) Eosin staining: Place the slices in a jar of eosin staining solution and stain for 3 to 5 minutes. The cytoplasm will turn pink.

[0211] (4) Dehydration and clearing: After washing the sections with tap water, dehydrate and clear them according to the following steps: 75% alcohol, 2 min; 85% alcohol, 2 min; 95% alcohol, 2 min; anhydrous ethanol, 2 min; anhydrous ethanol, 2 min; 1 / 2 xylene, 5 min; xylene, 5 min; xylene, 5 min.

[0212] (5) Sealing: Add a drop of transparent nail polish to the slice and slowly cover the slice with a coverslip from one side to avoid bubbles. After sealing, observe the tissue and cell morphology under a microscope.

[0213] 8. Masson staining experiment

[0214] (1) Hematoxylin staining: Using Masson staining reagent, add hematoxylin staining solution to the hydrated tissue sections to cover the tissue. After staining for 5 minutes, remove the staining solution and wash with tap water. The cell nuclei in the sections will be stained blue.

[0215] (2) Differentiation and blueing: Acidic differentiation solution was dripped onto the slices for 30 seconds, and tap water was used for blueing for 10 minutes.

[0216] (3) Ponceau-Acid Fuchsin Staining: Immerse the sections in Ponceau-Acid Fuchsin staining solution for 5-10 minutes, then remove and rinse with tap water for 5-10 seconds. Subsequently, add phosphomolybdic acid differentiation solution, differentiate for 2 minutes, and then remove.

[0217] (4) Aniline blue staining: Place the sections directly into aniline blue staining solution for 1 minute, then remove and rinse with tap water for 5-10 seconds. Then, add acidic differentiation solution and differentiate for 1 minute before removing.

[0218] (5) Dehydration and clearing: Dehydrate and clear the sections according to step (4) in “7. Hematoxylin-eosin staining experiment”.

[0219] (6) Sealing: Slowly cover the slice with a coverslip from one side. After sealing, observe the tissue and cell morphology under a microscope. Blue represents cell nuclei and collagen fibers, and red represents muscle fibers and red blood cells.

[0220] 9. Tissue Immunofluorescence (IF)

[0221] (1) Antigen retrieval: Place the hydrated skin tissue sections in a microwave-boiled antigen retrieval solution for 3 minutes, allow to cool naturally for 7 minutes, and repeat the boiling and cooling process three times. Rinse the sections with 1× PBS for 5 minutes.

[0222] (2) Permeabilization: When detecting intracellular antigens, permeabilization treatment is performed. Place the slices in 0.3% Triton X-100 solution (Table 3.7) for 30-60 minutes, and then rinse with PBS for 5 minutes.

[0223] (3) Inactivation of endogenous catalase: 3% catalase was added to the slices and incubated for 25 min, and then rinsed with PBS for 5 min.

[0224] (4) Blocking: Add blocking solution and remove it after 1-2 hours at room temperature.

[0225] (5) Incubation with primary antibody: Dilute the primary antibody to an appropriate concentration using blocking solution according to the antibody instructions. Add the antibody to the sections, covering the tissue, and place in a humidified chamber. Incubate at 4°C overnight, then rinse with 1× PBS for 5 minutes.

[0226] (6) Incubation with fluorescent secondary antibody: Dilute the fluorescent secondary antibody with 1× PBS at a ratio of 1:1000, add it onto the slice, incubate at room temperature in the dark for 1-2 hours, and then rinse with 1× PBS for 5 minutes.

[0227] (7) Nuclear counterstaining: Dilute DAPI at 1:1000 with 1×PBS and add it onto the slice. Incubate at room temperature in the dark for 5-10 minutes, then rinse with 1×PBS for 3 minutes.

[0228] (8) Sealing: Slowly cover the slice with a coverslip from one side and then observe under a fluorescence microscope.

[0229] 10. Western Blot

[0230] (1) Preparation of protein samples

[0231] ① Lyse tissue: Wash fresh skin tissue with 1× PBS, grind into powder with liquid nitrogen, collect into a centrifuge tube, add appropriate amount of lysis buffer (RIPA:PMSF:phosphatase inhibitor prepared at a ratio of 100:1:1 before use), mix well, place on ice, and lyse for 1-2 hours.

[0232] ② Protein concentration detection: After lysis, centrifuge at 4°C, 12000 rpm, 10 min, collect the supernatant, and use the BCA protein quantification kit to detect the protein concentration.

[0233] ③ Protein denaturation and sample storage: Calculate the volume of protein supernatant, 5× SDA-PAGE loading buffer, and distilled water required for each group based on the protein density of 3ug / ul in the protein sample denaturation system. Add them to the PCR tube, mix well, and place in a PCR instrument at 98°C for 10 minutes. After cooling, the protein sample should be divided into aliquots and stored at -80°C to avoid repeated freezing and thawing.

[0234] (2) SDS-PAGE gel electrophoresis

[0235] Prepare the gel according to the SDS-PAGE gel kit instructions. Add 10 μl of Protein Maker and each protein sample to the gel well of the electrophoresis tank. Turn on the electrophoresis instrument and set the constant current to 12 mA / 30 min, then to 18 mA until the end of the electrophoresis.

[0236] (3) Transfer

[0237] Soak the PVDF membrane in methanol for a few seconds to activate it, then place it in transfer buffer. Then, place the bottom filter paper, PVDF membrane, protein gel, and top filter paper in the transfer box in that order. Once the transfer is complete, set the transfer machine to 25V, 1.0mA, and 30min.

[0238] (4) Closed

[0239] The PVDF membrane was removed, placed in 5% BSA, and blocked on a shaker for 2 h.

[0240] (5) Incubation with primary antibody

[0241] According to the antibody instructions, the primary antibody was diluted with 5% BSA to a working concentration, added to the PVDF membrane and incubated on a shaker at 4°C for 8-12 hours. After recovery, it was washed with 1× TBST for 5 minutes.

[0242] (6) Incubation with secondary antibody

[0243] The secondary antibody was diluted to an appropriate concentration in 1×TBST, incubated with the PVDF membrane on a shaker at room temperature for 2 h, the secondary antibody solution was discarded, and the membrane was washed with 1×TBST for 5 min, and repeated 3 times.

[0244] (7) Color development

[0245] Prepare the color development solution according to the kit instructions, and detect protein expression using a chemiluminescence imaging system.

[0246] 3. Results and Analysis

[0247] (1) Construction and evaluation of diabetic foot ulcer (DFU) rat model

[0248] In this example, a diabetic foot ulcer rat model was constructed according to the experimental process. Figure 3 As shown in Figure -A, type 2 diabetes was first induced by high-fat feeding combined with multiple injections of small doses of streptozotocin (STZ). Foot ulcer wounds were created in the SD rats in which type 2 diabetes was successfully induced to construct a type 2 diabetic foot ulcer (DFU) rat model, and then the rats were randomly divided into groups for subsequent treatment studies.

[0249] In order to analyze whether the DFU rat model is successfully established, it is important to evaluate the diabetic symptoms of the DFU model in each group using the normal group rats as a control. First, the body weight of the rats was monitored after four weeks of high-fat feeding. Figure 3 As shown in Figure 2-B, the weight of rats in the DFU model group increased significantly after high-fat feeding compared with the normal group, and decreased significantly after STZ injection. Secondly, the detection of fasting blood glucose, glucose metabolism and insulin resistance levels is the most direct and effective method to assess the symptoms of diabetes. Figure 3 As shown in Figure 3-C, after STZ injection, the fasting blood glucose levels of the DFU model rats in each group increased significantly and were higher than 11.1 mM for more than one week. Figure 3 As shown in -D, the blood glucose rise rate of each group of DFU model rats after glucose injection was significantly lower than that of the normal group, and the blood glucose at each time point was significantly higher than that of the normal group, and the glucose metabolism capacity was significantly weakened. Figure 3 -E, the insulin resistance index of the DFU model rats in each group was significantly higher than 2.5, showing obvious insulin resistance. In addition, the results of rat water intake and mouse litter weight are as follows Figure 3 -F~ Figure 3 As shown in Figure 5-G, compared with the rats in the normal group, the water intake and urine output of the DFU model rats in each group increased significantly.

[0250] In summary, compared with the normal control group, all DFU model rats developed typical diabetic symptoms after STZ injection, including weight loss, hyperglycemia, impaired glucose metabolism, insulin resistance, polydipsia, and polyuria. They also exhibited decreased activity, hair loss, and delayed wound healing during the subsequent experimental course, consistent with the pathogenesis and pathological symptoms of diabetic foot. Therefore, the diabetic foot ulcer rat model established in this study met the experimental requirements of this study. Furthermore, metformin significantly reduced diabetes-related markers in DFU rats, demonstrating its synergistic potential when combined with hUC-MSCs to improve systemic metabolic status.

[0251] (2) hUC-MSCs and their combination with Met promote wound closure

[0252] The wound healing process mainly includes four overlapping continuous stages: hemostasis, acute inflammation (4-6 days), proliferation (4-21 days) and remodeling (21 days-2 years). In order to explore the wound healing process of DFU rats, we recorded typical wound images of each group at different time points. The results are shown in Figure 2. Figure 4-A shows that the rats in the normal group showed acute inflammation on the 3rd and 7th days. Healthy and stable red granulation tissue appeared around the wound from the 7th day, and the epithelium rapidly and continuously proliferated to cover the entire wound surface to reduce the wound surface area. The wound contraction was significantly accelerated, and it was almost completely closed on the 14th day. It was completely healed on the 19th day without obvious scars. The wound surface of the DFU model rats showed a dark yellow-red color on the 7th day, accompanied by persistent signs of inflammation. The granulation tissue of the wound filled and proliferated slowly, the formed epithelial tissue was unstable, the skin around the wound was swollen, and ulcers were easy to form during the wound care process. The quantitative analysis of the wound closure rate on the 19th day was about 60%, and the wound healing was significantly inhibited. The overall pathological state of the chronic wound was shown, which shows that the present invention successfully constructed the DFU model rat.

[0253] To investigate the effects of hUC-MSCs and their combination with Met on wound healing in rats with a DFU model, the present invention randomly divided the DFU rats into four groups. A PBS-injected control group was used. The DFU rats were treated with Met via oral gavage, subcutaneous intramuscular injection of hUC-MSCs into the wound, and hUC-MSCs combined with metformin. None of the DFU rats injected with hUC-MSCs developed skin tumors near the injection site, and no significant adverse reactions or transplant rejection occurred during the 19-day treatment period.

[0254] The typical wound images of each treatment group were recorded and a wound tracing model was constructed, e.g. Figure 4 -A and Figure 4 -B shows that the wound area of ​​each treatment group was significantly smaller than that of the control group. Although scabs were formed in the control group and the Met group on the 14th and 19th days, they were accompanied by inflammation and slow proliferation of granulation tissue and re-epithelialization, and the ulcer area was still large. The MSC treatment group and the combined treatment group showed significantly smaller wound areas at each observation time point (i.e., the 3rd, 7th, 14th and 19th days after treatment), the inflammatory symptoms were significantly weakened, the red granulation tissue around the wound was accelerated and gradually epithelialized, the wound contraction was significantly accelerated, and it was almost completely healed on the 19th day. The wound area was further quantified to analyze the wound closure rate, and the results are shown in Figure 5. Figure 4 As shown in Figure 3-C, each treatment group promoted wound closure to varying degrees. The MSC-treated and combined treatment groups exhibited the highest wound closure rates, reaching approximately 80% on day 14 and over 90% on day 19, demonstrating the most significant effect. Notably, the wound closure rate in the combined treatment group approached that of the normal control group, indicating that Met further promoted the efficacy of hUC-MSCs, exerting a synergistic effect.

[0255] (3) hUC-MSCs and their combination with Met promote wound re-epithelialization and collagen deposition

[0256] Healing of diabetic foot ulcers is defined as complete epithelialization of the ulcer site. Re-epithelialization and tissue repair begin during the proliferative phase of the skin healing process, typically manifesting as thinning and maturation of the thickened surface layer of the skin. The skin is composed, from the outside in, of the epidermis, dermis, and subcutaneous tissue. The epidermis is composed of squamous, keratinized epithelial cells, while the dermis is composed of collagen fibers and reticular fibers. It is divided into a superficial layer and a deeper reticular layer, which are home to numerous blood vessels, nerves, hair follicles, sweat glands, and sebaceous glands. It is rich in extracellular matrix (ECM), vasculature, and mechanoreceptors.

[0257] In order to gain a deeper understanding of the tissue repair details of diabetic foot ulcer wound re-epithelialization from a microscopic perspective, the present invention conducted a histological evaluation of foot skin samples after treatment. The results of H&E staining sections are as follows: Figure 5 -As shown in A, compared with the normal feet, the epidermis of DFU rats was interrupted on both sides and the arrangement of keratinization was disordered, and wound healing was delayed. On the 19th day, the wounds of all treatment groups showed varying degrees of epidermal re-epithelialization structure and stratum corneum coverage. Compared with the control group, the epidermis of the Met treatment group, MSC treatment group and combined treatment group was intact and continuously arranged, and the dermal proliferation thickness increased significantly. In particular, the increase in epithelial thickness in the MSC treatment group and the combined treatment group was the most significant, and the superficial dermis area around the center of the wound was significantly thinned, promoting skin healing and maturation. Further quantitative analysis of the wound center area of ​​each group was performed, and the results are shown in the figure below. Figure 5 As shown in -B, the width of the wound center area in the MSC treatment group and the combined treatment group was significantly reduced, and the dermal new granulation tissue increased significantly. Skin appendages such as hair follicles, sebaceous glands, and sweat glands can be used to evaluate the integrity of regenerated skin tissue. The number of hair follicles was further quantified and the results were as follows Figure 5 -C shows that there were almost no new hair follicles in the wound healing area in the control group and the Met treatment group, while the number of hair follicles in the wound healing area in the MSC treatment group and the combined treatment group was significantly higher, indicating that subcutaneous transplantation of hUC-MSCs promoted the formation of skin appendages such as hair follicles, and Met played a certain synergistic role to show the best skin function recovery.

[0258] Collagen deposition in the reticular layer of the wound dermis plays an important role in wound contraction, scar formation and ECM remodeling. Figure 6 As shown in -A, compared with the control group, the amount of blue collagen fiber deposition in each DFU rat treatment group was significantly increased, and the amount of red muscle fiber was significantly reduced. Among them, the collagen fiber deposition area in the MSC treatment group and the combined treatment group was the largest, and the muscle fiber area was the smallest. Further quantification of the collagen deposition area showed the following results: Figure 6As shown in -B, the collagen deposition area of ​​the MSC treatment group and the combined treatment group reached approximately 60% and 66% on the 19th day, respectively, which is close to that of the normal group (approximately 70%) and significantly higher than that of the control group (approximately 27%). This indicates that the MSC treatment group significantly promotes the deposition of new collagen and Met promotes the effect of stem cell therapy. Subsequently, in order to evaluate the difference in matrix remodeling, the invention conducted an immunoblotting experiment, and the results are shown in Figure 2. Figure 7 As shown, compared with the control group, wound collagen Col I, Col III, and the Col III / I ratio increased to varying degrees in each treatment group of DFU rats on day 19, while matrix metalloproteinases (MMP2, MMP3, and MMP9) decreased to varying degrees. The most significant differences were observed in the MSC-treated and combined treatment groups, respectively. This suggests that subcutaneous transplantation of hUC-MSCs significantly increased type I collagen deposition and promoted type III collagen production, contributing to scarless wound healing. The ratio of type III / type I collagen fibers was significantly increased, inhibiting fibrotic scar formation and accelerating wound healing and tissue repair. Furthermore, in the late stages of wound healing, matrix metalloproteinase expression was significantly reduced, reducing the degradation of extracellular matrix components, consistent with the dynamic regulation of wound healing. In the combined treatment group, Met exerted a synergistic effect, significantly accelerating collagen deposition and matrix maturation, promoting optimal tissue repair outcomes and progression.

[0259] (4) hUC-MSCs and their combination with Met promote wound cell proliferation and cytokine expression

[0260] The proliferation stage is a key dynamic regulation in the wound healing process. It is enhanced in the early stage of healing and weakened in the later stage. It involves the activation, proliferation, migration of various cells and their interactions with various molecular mediators, which jointly promote re-epithelialization and matrix deposition, angiogenesis, and inflammation resolution. First, the cell proliferation of DFU rats in the proliferation stage of wound healing on the 14th day of treatment was detected by immunohistofluorescence staining with anti-Ki67 antibodies. The results are as follows Figure 8 As shown in -A, the Ki-67 positive cells in the MSC treatment group and the combined treatment group were significantly more, and all were distributed in the basal layer of the wound. Figure 8 -B shows that the percentage of Ki-67 positive cells in each treatment group was significantly higher than that in the control group to varying degrees, among which the statistical differences between the MSC treatment group and the combined treatment group were the most significant. Secondly, CD31 and CD34, α-SMA, FSP1, K14, CD163, and CD86 were used as markers of vascular endothelial cells, vascular smooth muscle cells, fibroblasts, keratinocytes, M2 anti-inflammatory macrophages, and M1 pro-inflammatory macrophages, respectively. The expression levels of markers of important cells in the wound healing stage were detected by WB experiments. The results are shown in the figure below. Figure 8-C, the expression levels of CD31 and CD34, α-SMA, FSP1, K14, and CD163 in the MSC treatment group and the combined treatment group were significantly higher than those in the control group and the Met treatment group, and the expression level of CD86 was significantly lower than that in the control group and the Met treatment group. At the same time, the expression levels of important molecular mediators in the wound healing stage were detected by WB experiments. The results are shown in Figure 2. Figure 8 As shown in Figure 3-D, the expression levels of various cytokines and mediators that accelerate the wound healing process were significantly insufficient in the DFU rat control group. However, the expression levels of vascular endothelial growth factor-A (VEGFA), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), and interleukin-10 (IL-10) in the MSC treatment group and the combined treatment group were significantly higher than those in the control group and the Met treatment group, while the expression levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were significantly lower than those in the control group and the Met treatment group, indicating that Met played a synergistic role.

[0261] The proliferative phase of wound healing is typically characterized by robust angiogenesis and the appearance of multiple pro-angiogenic factors. High expression levels of the endothelial cell markers CD31 and CD34, the vascular smooth muscle cell marker α-SMA, and the related angiogenic factors VEGFA, TGF-β, and bFGF were observed in the MSC-treated and combined-therapy groups, indicating enhanced angiogenesis in the granulation tissue during the proliferative phase of wound healing in these two groups, whereas angiogenesis was blocked in the control group. The re-epithelialization phase requires the proliferation and migration of keratinocytes to reorganize the epidermis, as well as fibroblast-mediated collagen matrix reconstruction. High expression levels of the keratinocyte marker K14, the fibroblast marker FSP1, and the related cytokines TGF-β and bFGF in the MSC-treated and combined-therapy groups are consistent with previous experimental results showing that stem cell transplantation can significantly increase the re-epithelialization of diabetic foot ulcer wounds. Furthermore, the proliferative phase of wound healing is accompanied by the resolution of acute inflammation, as evidenced by the initial conversion of monocytes into proinflammatory M1 macrophages at the site of injury. Subsequently, as the inflammatory response progresses, they repolarize into anti-inflammatory M2 macrophages under the influence of factors such as TGF-β and IL-10. Typically, macrophage polarization is blocked in diabetic foot ulcer wounds, leading to prolonged stagnation in the chronic inflammatory microenvironment and delayed wound healing. Compared with the control and Met groups, the MSC-treated and combined treatment groups showed elevated expression of CD163, a marker for anti-inflammatory M2 macrophages, and related cytokines TGF-β and IL-10, while expression of CD86, a marker for proinflammatory M1 macrophages, and related cytokines TNF-α and IL-1β was reduced. This suggests that subcutaneous stem cell transplantation promotes wound healing in DFU rats, promotes macrophage polarization from M1 to M2, and alleviates the chronic inflammatory microenvironment, with Met acting as a synergistic agent, consistent with previous experimental results.

[0262] Example 3. Effects of hUC-MSCs and their combination with Met on angiogenesis in DFU rat wounds

[0263] 1. Experimental Materials

[0264] 1. Cells and tissues

[0265] (1) Human umbilical cord mesenchymal stem cells, see Example 1.

[0266] (2) Skin tissue sections of rat feet with diabetic foot ulcers, see Example 2.

[0267] 2. Experimental solution formula

[0268] (1) The solution formula related to hUC-MSCs cell culture experiment is shown in Example 1.

[0269] (2) For the solution formulas related to protein immunoblotting and tissue immunofluorescence experiments, see Example 2.

[0270] (3) 6 mM metformin stock solution: Take 19.87 mg of metformin and 20 ml of sterile water, prepare 6 mM metformin stock solution in a clean bench, mix thoroughly and divide into aliquots, keep at -20 °C away from light, avoid repeated freezing and thawing, and dilute to the target concentration with basal culture medium before use.

[0271] (4) 1 M glucose stock solution: Take 9.91 g of glucose and 50 ml of basal culture medium to prepare 1 M glucose stock solution in a clean bench. Mix thoroughly and then divide into aliquots. Keep at 4°C to avoid repeated freezing and thawing. Dilute with basal culture medium to the desired concentration before use.

[0272] 2. Experimental Methods

[0273] 1. MTT assay

[0274] (1) Cell seeding: Digest and wash hUC-MSCs and prepare a single-cell suspension. Seed 1000-2000 cells into a 96-well plate, 200 μl per well, repeat 3 wells, and place in an incubator.

[0275] (2) Drug grouping: For glucose half-inhibitory concentration (IC50) detection, the groups were as follows: 0mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 70mM, 90mM, 110mM, 130mM, all treated for 3 days. For metformin growth activity detection, the groups were as follows: 0mM, 0.05mM, 0.1mM, 0.3mM, 0.5mM, 1mM, 3mM, all treated for 3 days. For hUC-MSC growth curve detection, the groups were as follows: PBS group, 30mM glucose group, 30mM glucose combined with 0.3mM metformin treatment group, treated for 1, 2, 3, 4, 5, 6, 7 days in sequence. Use basal culture medium to gradiently dilute 1M glucose stock solution and 6mM metformin stock solution to the above target concentrations. Take 200ul of each aliquot and transfer to a 96-well plate. Repeat 3 times and then return to the cell culture incubator for culture.

[0276] (3) MTT incubation: After the incubation time point, add 20 μl of MTT solution (5 mg / ml) to each well, gently shake to mix, and return to the cell culture incubator for approximately 4 hours. Then carefully remove the supernatant from the wells, add 200 μl of DMSO to each well, and shake to mix.

[0277] (4) Absorbance determination: The absorbance value (OD value) at a wavelength of 560 nm was measured in a microplate reader, and the data were analyzed and the growth activity was calculated: survival rate (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0278] 2. Western Blot

[0279] hUC-MSCs were treated for 3 days in the following groups: PBS, 30 mM glucose, and 30 mM glucose plus 0.3 mM metformin. Total cells were collected and centrifuged to obtain a cell pellet. Subsequently, the Western Blot protocol described in Example 2 was followed, with approximately 200 μl of lysis buffer added for subsequent procedures.

[0280] 3. Tissue Immunofluorescence (IF)

[0281] The specific steps are the same as those in the tissue immunofluorescence experiment in Example 2.

[0282] 3. Results and Analysis

[0283] (1) hUC-MSCs and their combination with Met promote the formation of wound vascular structures

[0284] Angiogenesis is a key link in the wound healing process, providing the necessary oxygen and nutritional support for wound repair and cells. Therefore, promoting angiogenesis and solving the problem of microvascular dysfunction are important directions for treating diabetic foot ulcer wound healing. The present invention detected the angiogenesis regulation of DFU rats on the 7th and 14th days after treatment, and the results were as follows through the immunohistofluorescence co-staining experiment of CD31 (vascular endothelial cell marker) and α-SMA (vascular smooth muscle cell marker). Figure 9 -A and Figure 9 -As shown in B, the density, number, and cross-section of the vascular structure of DFU rats were much lower than those of the normal group on the 7th and 14th days, indicating that abnormal damage occurred in the construction and maintenance of their functional special vascular system. On the 7th day, the MSC treatment group and the combined treatment group clearly showed the extensive formation of dense microvascular systems, and the density of small blood vessels was much higher than that of the control group and the Met treatment group; in some areas, there were CD31 cells but no α-SMA cells, indicating the generation of microcapillaries. On the 14th day, the double-positive lumen structures in the MSC treatment group and the combined treatment group were significantly larger, gradually maturing into stable blood vessels, and the generation of immature small blood vessels gradually decreased or degenerated. The number and area of ​​vascular structures in immunohistofluorescence were further quantified, and the results are shown in the figure below. Figure 9 -C~ Figure 9 -E, compared with the control group, the number of CD31-positive blood vessels and CD31 and α-SMA double-positive blood vessels in the MSC treatment group and the combined treatment group increased significantly on the 7th day, and the number of angiogenesis decreased on the 14th day, but was still significantly higher than that in the control group and the Met treatment group; the vascular structure area in the MSC treatment group and the combined treatment group had a small significant difference from that in the normal group on the 7th day, but the vascular ring area on the 14th day was significantly higher than that in the control group and the Met treatment group, and the vascular structure continued to strengthen.

[0285] (2) hUC-MSCs and their combination with Met promote the expression of wound angiogenesis-related proteins

[0286] In order to further clarify whether the regulation of angiogenesis and vascular function are effectively exerted, the present invention detects the key signaling molecules of angiogenesis in DFU rat wounds. The VEGF / VEGFR signaling pathway is one of the most critical pro-angiogenesis signaling pathways. The immunohistofluorescence staining experimental results of VEGFA antibodies and their quantification results are shown in Figure 2. Figure 10 -A and Figure 10 -B shows that on the 14th day after treatment, the vascular endothelial growth factor (VEGFA) in the DFU rat wound of the control group was still very low; the expression and percentage of VEGFA in the MSC treatment group and the combined treatment group were significantly higher. Figure 10-C shows that on the 14th day after treatment, the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2), and endothelial nitric oxide synthase (eNOS) in the MSC treatment group and the combined treatment group were significantly higher than those in the control group DFU rats, and the vascular endothelial cells in the wound environment were in a phosphorylated activated state; the expression levels of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were higher than those in the control group, which helped cell migration and was recruited to promote angiogenesis, while the p-VEGFR2, eNOS, MMP2 and MMP9 factors in the normal group rats were only lower than those in the MSC treatment group and the combined treatment group. It is worth noting that, combined with the previous wound healing and vascular structure generation analysis, this suggests that angiogenesis in the normal group rats was enhanced and gradually matured on the 14th day, and angiogenesis was weakened. At the same time, the previous immunoblotting results ( Figure 7 ) showed that on the 19th day, the MMP2 and MMP9 in the normal group, MSC treatment group and combined treatment group were significantly lower than those in the control group and Met treatment group, which suggested that the vascular structure of hUC-MSCs and its combined Met matured and angiogenesis weakened on the 19th day, which was consistent with the angiogenesis regulation direction of normal rat wounds.

[0287] (3) hUC-MSCs remain in the wound dermis

[0288] The therapeutic effect of cell transplantation therapy is still limited by the low cell retention rate, survival rate and implantation rate. To further explore the retention and implantation of subcutaneously transplanted hUC-MSCs in DFU rat wounds, the present invention detected the presence of hUC-MSCs cells with proliferation activity in skin tissue on the 3rd and 7th days after transplantation. The results of K14 and EdU fluorescence co-staining are shown in Figure 2. Figure 11 As shown in Figure 2-A, EdU-positive (green) hUC-MSCs in both the MSC-treated and combined-treatment groups were primarily distributed beneath the superficial dermis on day 3. By day 7, the number of cells had significantly decreased and was primarily distributed in the deep reticular layer of the dermis, contributing to wound healing. This is consistent with previous studies showing that diabetes-induced hyperglycemia damages mesenchymal stem cells and inhibits their survival. Compared to the MSC-treated group, the combined-treatment group retained the most hUC-MSCs at all time points, suggesting that Met may prolong cell retention.

[0289] In order to verify the effects of high sugar environment and Met on hUC-MSCs cells, the present invention conducted an in vitro MTT experiment. The results are as follows Figure 11As shown in Figure 3-B, under high glucose concentration, hUC-MSCs cell viability decreased with increasing glucose concentration; the half-inhibitory concentration of glucose on hUC-MSCs cells on day 3 was 94.87 mM. In the present invention, the random blood glucose level of DFU rats was no higher than 35 mM, which was far below the half-inhibitory concentration, indicating that hUC-MSCs cell survival was inhibited by the high glucose microenvironment, but did not affect the number of transplanted cells (approximately 2×10 6 cells / kg) to achieve cell survival and retention of therapeutic effects. Combined with the actual blood sugar level of DUF rat model, the present invention selected 30mM glucose concentration for subsequent experiments. The effect of Met on hUC-MSCs cell viability was tested according to the pharmacological dose of Met. The results are as follows Figure 11 As shown in -C, Met at 0.1mM and 0.3mM can significantly promote the viability of hUC-MSCs cells, and gradually inhibit the viability of hUC-MSCs cells with increasing concentration. Met 0.3mM was selected for subsequent experiments. The effect of Met on the proliferation activity of hUC-MSCs in high glucose environment was further tested. The results are shown in Figure 11 -D shows that within 7 days, Met can significantly enhance and restore the hyperglycemia-induced inhibition of hUC-MSCs cell proliferation activity, which is consistent with the previous experimental results.

[0290] (4) Paracrine function of hUC-MSCs in the vascular structure of the wound dermis

[0291] In order to further study the function of hUC-MSCs cells in the basal layer of DFU rat wounds, the present invention conducted fluorescence co-staining experiments of CD31, α-SMA and EdU on the DFU rat combined treatment group on the 7th day. Figure 12 -A and Figure 12 -B shows that EdU and CD31 positive cells, as well as EdU and α-SMA positive cells, can be detected in the newly generated blood vessels, and none of them are co-localized. This indicates that under the combined treatment of Met, hUC-MSCs colonize in the vascular structure of the deeper reticular layer of the dermis to promote angiogenesis, but there is no sign of differentiation into vascular endothelial cells and vascular smooth muscle cells. Therefore, it is speculated that subcutaneous muscle transplantation of hUC-MSCs may promote angiogenesis and healing of diabetic wounds through a paracrine mechanism at the wound site. In order to further detect the paracrine function of hUC-MSCs and its combination with Met in a high glucose environment, we performed immunoblotting experiments, and the results are shown in the following figure. Figure 12 -C and Figure 12As shown in Figure 3-D, hUC-MSCs highly expressed factors such as VEGFA, TGFβ-1, bFGF, and IL-10, which were significantly reduced after high glucose treatment but significantly restored after Met treatment. This indicates that the hUC-MSCs extracted in this study have strong paracrine capacity, which can provide a large amount of exogenous angiogenic cytokines for the angiogenesis process in DFU rats to promote wound healing. Moreover, the pharmacodynamic concentration of Met can significantly restore the paracrine capacity of hUC-MSCs cells that is damaged by the high glucose environment, thereby exerting a combined therapeutic effect of promoting angiogenesis.

[0292] Example 4. Effects of hUC-MSCs culture supernatant (MSC-CM) and its combination with Met on vascular endothelial cells

[0293] 1. Experimental Materials

[0294] 1. Cells

[0295] (1) Human umbilical cord mesenchymal stem cells, see Example 1.

[0296] (2) Human umbilical vein endothelial cells (HUVEC)

[0297] Cultivate according to specifications to ensure the accuracy of subsequent experiments.

[0298] 2. Experimental solution formula

[0299] (1) The solution formulas for hUC-MSCs cell culture experiments and EdU experiments are shown in Example 1.

[0300] (2) The relevant solution formulas used in MTT, plate cloning, cell scratch and migration experiments are shown in Example 3.

[0301] (3) HUVEC cell culture medium formula: DMEM high glucose basal medium 45 ml, fetal bovine serum 5 ml, P / S 500 ul.

[0302] (4) HUVEC cell high glucose culture medium (supplemented with 30 mM glucose) formula: DMEM high glucose basal medium 45 ml, fetal bovine serum 5 ml, P / S 500 ul, 1 M glucose solution 1.55 ml.

[0303] (5) 1% serum cell culture medium formula: MEM-Alpha basal medium 45ml, fetal bovine serum 500ul, P / S 500ul.

[0304] 2. Experimental Methods

[0305] 1. MTT assay

[0306] (1) Cell inoculation and drug grouping treatment: Inoculate 1000-2000 cells into a 96-well plate, 200ul per well, repeat 3 wells, and culture overnight before performing the following drug grouping treatment. For glucose half-inhibitory concentration (IC50) detection, grouping is as follows: 0mM, 5mM, 10mM, 20mM, 30mM, 50mM, 70mM, 90mM, all treated for 3 days. For metformin growth activity detection, grouping is as follows: 0mM, 0.05mM, 0.1mM, 0.3mM, 0.5mM, 1mM, 3mM, all treated for 3 days. Use basal culture medium to gradiently dilute 1M glucose stock solution and 6mM metformin stock solution to the above target concentrations, take 200ul according to grouping and transfer to a 96-well plate, repeat 3 times and continue to culture in the cell culture incubator.

[0307] (2) MTT incubation and absorbance determination: After culturing to the time point, the subsequent steps of the MTT experiment (for specific steps, refer to Example 3) are followed.

[0308] 2. Construction of HUVEC high-glucose culture model and drug intervention treatment

[0309] (1) HUVEC cell culture: HUVEC cells were cultured in normal and high-glucose medium. The high-glucose culture group was subcultured with HUVEC cells in high-glucose medium for 2 weeks to construct a high-glucose treatment (HG) cell model, and then the cells were treated with drug intervention groups.

[0310] (2) Preparation of serum-free culture supernatant: When hUC-MSCs cells reached approximately 80% confluency, the culture medium was replaced with one containing or containing 1% serum. The cells were cultured until fully confluent. The culture supernatant was collected and centrifuged at 1200 rpm for 5 min. The supernatant was transferred to new sterile centrifuge tubes and stored at 4°C until further use. Long-term storage was at -80°C. The cell culture supernatant of HUVEC cells was collected in the same manner and aliquoted for storage.

[0311] (3) Drug intervention groups: normal group, high glucose model group, metformin-treated group, stem cell supernatant-treated group, and stem cell supernatant combined with metformin-treated group, for a total of 5 groups. The culture medium for each group was prepared according to Table 6 and used for subsequent cell EdU experiments, plate cloning, wound wound healing, migration, angiogenesis experiments, and cell pellet sample collection.

[0312] Table 6. Drug intervention culture medium

[0313]

[0314] 3. EdU experiment

[0315] (1) Cell inoculation and drug grouping treatment: HUVEC cells cultured in normal and high glucose conditions were inoculated into 24-well plates at approximately 2×104 After the cells were cultured overnight, the culture medium was removed and the drug intervention culture medium was replaced according to the experimental groups in Table 6, and the culture was continued for 48 hours.

[0316] (2) After 48 h of drug intervention and culture, cell detection was performed according to the subsequent steps of the EdU experiment (same as Example 1).

[0317] 4. Plate cloning experiment

[0318] (1) Cell inoculation and drug grouping: HUVEC cells cultured in normal and high glucose conditions were digested, washed, and counted, then inoculated into 12-well plates with approximately 800 cells per well. After overnight adherence culture, the culture medium was aspirated and the drug intervention culture medium was replaced according to the experimental groups in Table 6. Culture was continued until clones were formed, which was approximately 7-10 days. During this period, the culture medium was changed according to the experimental groups as appropriate.

[0319] (2) Fixation and staining: After the incubation period, wash with 1×PBS; add 4% PFA solution to fix for 15-30 minutes, remove, and wash with 1×PBS; add crystal violet staining solution to stain for 15-30 minutes, remove, and wash with 1×PBS.

[0320] (3) Observation and analysis of results: Scan the stained culture plates with a scanner, and count and perform statistical analysis.

[0321] 5. Cell scratching

[0322] (1) Cell inoculation and scratching: HUVEC cells cultured in normal or high glucose conditions were inoculated into 6-well plates. When the cell fusion was above 90%, a straight line of uniform thickness was vertically scratched in the middle of each well using a 10 μL pipette tip. The suspended cells were washed away with 1× PBS, and a picture of the cells was taken at 0 h after the scratching.

[0323] (2) Drug Intervention Grouping and Observation: Low-serum drug intervention culture medium was prepared according to the experimental groups in Table 6, with 500 μl of fetal bovine serum added to each group. Low-serum drug intervention culture medium was added to the wells of the plate according to the group, and observation and photography were performed 24 and 36 hours after the scratch.

[0324] (3) Observation and data analysis: The scratch width at different time points was measured using image analysis software, and the 36h cell migration rate (%) was calculated as (0h scratch width - 36h scratch width) / 0h scratch width × 100%.

[0325] 6. Cell migration assay

[0326] (1) Cell inoculation and drug intervention group treatment: HUVEC cells cultured normally and in high glucose were digested and washed to prepare single cell suspensions, counted, and the cell concentration was adjusted to approximately 3×10 5600ul of drug intervention culture medium was added to a 24-well plate according to the experimental group, and 200ul of cell suspension was added to the Transwell chamber and cultured for 24-48 hours.

[0327] (2) Fixation and staining: After the completion, remove the chamber, wipe the cells on the surface of the chamber membrane with a cotton swab, wash with 1×PBS and then transfer the chamber to a new plate well. Add 4% paraformaldehyde solution to fix for 15-30 minutes and then remove. Add 1×PBS to wash and then remove. Add an appropriate amount of crystal violet staining solution for about 15-30 minutes and then remove. Then wash the cells with 1×PBS to remove the staining solution.

[0328] (3) Observation and data analysis: Observe the stained cells on the lower surface of the chamber membrane under an inverted microscope, take pictures, and use image analysis software to randomly count the number of cells in several fields of view, and calculate the average value as the migration ability.

[0329] 7. Matrigel tube formation experiment

[0330] (1) Angiogenesis Matrigel Spreading: All procedures should be performed on an ice box in a clean bench, and all equipment should be pre-chilled. Add 70 μl of Matrigel to a 24-well plate, evenly covering the bottom of the wells, and incubate in an incubator for 30-60 minutes to allow it to solidify.

[0331] (2) Cell inoculation and drug intervention grouping: HUVEC cells cultured in normal and high glucose conditions were digested and washed, and then divided into 5 groups according to drug intervention and centrifuged. Cells were resuspended in the drug intervention culture medium of each group and inoculated with 1×10 5 Cells were plated in a 24-well plate, 500 μl per well, and gently placed in the incubator.

[0332] (3) Observation and result analysis: 6 h, 12 h, and 24 h after cell inoculation, the vascular-like structures were observed under an inverted microscope, and parameters such as the number of vascular branches, total length, and area enclosed by the vessels were analyzed using image analysis software.

[0333] 8. Protein immunoblotting

[0334] (1) Cell inoculation and drug grouping: HUVEC cells cultured in normal and high glucose conditions were digested, washed, counted, and inoculated into culture dishes. After adherence and overnight culture, the culture medium was removed. When the cell fusion rate was approximately 50%, the drug intervention culture medium was replaced according to the experimental groups in Table 6.

[0335] (2) After 48 h of drug intervention, the cells were tested according to the subsequent experimental steps of the immunoblotting experiment (same as in Example 2).

[0336] 3. Results and Analysis

[0337] (1) MSC-CM and its combination with Met promote HUVEC cell proliferation

[0338] The vascular endothelial cells of normal wounds are the main cells involved in the angiogenesis process. However, the high blood sugar environment in DFU patients will affect the survival and cell function of vascular endothelial cells to hinder angiogenesis. In order to further verify the effect of hUC-MSCs paracrine ability on angiogenesis in DFU rats, vascular endothelial cells were used as the research object to explore the role of hUC-MSCs culture supernatant (MSC-CM) and HUVEC cells in co-cultured survival and proliferation in a high glucose environment in vitro. First, the results of the in vitro MTT experiment preliminarily showed that at high glucose concentrations, the viability of HUVEC cells decreased with increasing glucose concentrations; the half-inhibitory concentration of glucose on HUVEC cells on the third day was 62.91mM, see for details. Figure 13 -A. Based on the actual blood glucose level of DUF rat model, this study selected 30mM glucose concentration for subsequent experiments. The effect of Met on HUVEC cell viability was investigated according to the pharmacological dose. The results are as follows Figure 13 As shown in Figure 2B, Met at 0.3 mM can significantly promote HUVEC cell viability.

[0339] This study continued to explore the effects of MSC-CM and Met on the proliferation of HUVEC in a high glucose environment in vitro through EdU detection and plate cloning experiments. Figure 13 -C and Figure 13 -D, the percentage of EdU positive cells in the MSC-CM treatment group and the combined treatment group was significantly higher than that in the control group and the Met group. Figure 13 -E and Figure 13 -F, the number of cell clones in the MSC-CM treatment group and the combined treatment group was significantly higher than that in the control group. Finally, immunoblotting experiments were performed, and the results were shown in Figure 13 As shown in Figure 3-G, the expression levels of the anti-apoptotic protein Bcl-2 in the MSC-CM and combined treatment groups were significantly higher than those in the control group, while the expression levels of the pro-apoptotic protein Bad were significantly lower than those in the control group. These results indicate that the culture supernatant of hUC-MSCs (MSC-CM) can significantly restore the proliferation capacity of HUVEC cells in a high-glucose environment, promote HUVEC cell proliferation, and alleviate their damage in a high-glucose environment. Met can further significantly promote the proliferation capacity of HUVEC through synergistic effects.

[0340] (2) MSC-CM and its combination with Met promote HUVEC cell migration

[0341] The process of angiogenesis requires that endothelial cells in the vascular system adjacent to the injured skin area be activated and induced to recruit to the wound site. This article continues to explore the role of HUVEC cell migration. First, the results of the cell scratch experiment are as follows Figure 14 -A and Figure 14 As shown in Figure 2B, the scratch healing ability of HUVEC cells was significantly inhibited within 36 hours in a high glucose environment, while the scratch healing migration rate of the MSC-CM treatment group and the combined treatment group was significantly higher than that of the control group and the Met treatment group, and the healing rate was significantly restored. Further quantitative analysis confirmed the above observations. Transwell experiment and its quantitative results are shown in Figure 2B. Figure 14 -C and Figure 14 As shown in Figure 3D, the number of HUVEC cells migrating in a high glucose environment within 36 hours was significantly reduced, and their migration ability was significantly inhibited. However, the number of cells migrating in the MSC-CM treatment group and the combined treatment group was significantly higher than that in the control group and the Met treatment group, and cell migration was significantly restored. Figure 14 As shown in Figure 3-E, compared with the HG control group, the MSC-CM and combined treatment groups showed a significant downregulation of E-cadherin expression, reducing adhesion to epithelial cells, and significantly upregulated the expression of proteins involved in promoting cell migration, such as N-cadherin, vimentin, matrix metalloproteinase 9 (MMP9), and matrix metalloproteinase 2 (MMP2). These results consistently indicate that the culture supernatant of hUC-MSCs (MSC-CM) can significantly restore the migration ability of HUVEC cells in a high-glucose environment, and Met plays a synergistic role.

[0342] (3) MSC-CM and its combination with Met promote HUVEC cell angiogenesis

[0343] The process of angiogenesis is that endothelial cells continuously aggregate to form a lumen structure, which then grows larger to form a mature vascular system, and then gradually weakens. Next, we continued to explore the effect of MSC-CM on angiogenesis of HUVEC cells in a high glucose environment by using the Matrigel tube formation method. The results of the Matrigel angiogenesis experiment are as follows Figure 15 As shown in Figure 2A, the formation of blood vessels in HUVEC cells was significantly inhibited in a high-glucose environment, while the number of microvascular structures in the MSC-CM and combined treatment groups was significantly greater than that in the control and Met treatment groups within 6 hours, and continued to mature over time. At 24 hours, the lumen structure was significantly larger than that in the control group. Further quantification of vascular nodes, lumen number, and total length was shown in Figure 2B. Figure 15 -B~ Figure 15As shown in -D, the number of vascular nodes, lumen and total length in the MSC-CM treatment group and the combined treatment group continued to decrease with the maturation of angiogenesis, and were significantly higher than those in the control group and the Met group at each time point. The VEGF / VEGFR signaling pathway is one of the most critical angiogenesis-promoting signaling pathways. The protein molecules regulating angiogenesis were detected by immunoblotting experiments. The results are shown in Figure 15 As shown in Figure 5-E, the expression levels of VEGFA, TGF-β, and p-VEGFR2 in the MSC-CM and combined treatment groups were significantly upregulated compared with the HG control group. These results consistently indicate that the culture supernatant of hUC-MSCs (MSC-CM) can significantly restore the angiogenesis ability of HUVEC cells in a high glucose environment, and Met plays a synergistic role.

[0344] Example 5. Effects of hUC-MSCs paracrine secretion on angiogenesis signaling pathways

[0345] 1. Experimental Materials

[0346] (1) Human umbilical cord mesenchymal stem cells, see Example 1.

[0347] (2) Human umbilical vein endothelial cells, see Example 4.

[0348] (2) Skin tissue sections of rat feet with diabetic foot ulcers, see Example 2.

[0349] 2. Experimental Methods

[0350] 1. Transcriptome Sequencing

[0351] (1) Cell seeding and drug treatment: HUVEC cells cultured in normal and high-glucose cultures were digested, washed, counted, and seeded into culture dishes. After overnight adherence, the culture medium was aspirated and drug treatment was performed when the cell confluence rate was approximately 50%. The experimental groups were divided into a normal control group, a high-glucose model group, and a stem cell supernatant-treated group. The drug-treated culture medium was prepared and replaced according to the experimental groupings in Table 6, and the cells were cultured for 48 h.

[0352] (2) Cell sample collection: After 48 h of drug intervention, the three groups of cells were collected and centrifuged. 500 μl of Trizol was added to the precipitate, and the cells were grouped, labeled, and temporarily stored at -80°C. They were then sent to a professional institution for sequencing as soon as possible.

[0353] (3) Transcription and sequencing: First, total RNA from each group of HUVECs was extracted and quantified. After quality confirmation, mRNA was obtained from the total RNA, sheared to fragments less than 300 bp using a fragmentation buffer, and then reverse transcribed into cDNA. A cDNA library was established and DNA sequencing was performed on a sequencing system.

[0354] (4) Bioinformatics analysis: DESeq2 software was first used to analyze the gene and transcript levels of the normal control group (Control), high glucose model group (HG), and stem cell supernatant treatment group (MSC-CM) in the RNAseq dataset; differentially expressed genes (DEGs) were obtained based on |log2 FC| ≥ threshold 0.585 and P correction < 0.05; finally, KEGG and GO pathway enrichment analysis was performed in R language software using the database.

[0355] 2. Protein immunoblotting

[0356] (1) Cell inoculation and drug grouping: HUVEC cells cultured in normal and high glucose conditions were digested, washed, counted, and inoculated into culture dishes. After adherence and overnight culture, the culture medium was removed. When the cell fusion rate was approximately 50%, the drug intervention culture medium was replaced according to the experimental groups in Table 6.

[0357] (2) After 48 hours of drug intervention and culture, detection was performed according to the subsequent steps of the immunoblotting experiment (same as Example 2).

[0358] 3. Immunohistochemistry (IHC)

[0359] (1) The hydrated skin tissue sections were subjected to antigen retrieval, permeabilization, catalase inactivation, and blocking steps according to the IF experimental procedure (same as Example 2).

[0360] (2) Primary Antibody Incubation: The primary antibody was diluted to a working concentration in blocking solution (i.e., the immunohistochemistry blocking solution in Example 2), added dropwise to the sections, incubated at 4°C overnight, and then rinsed with 1× PBS for 30 min. Reaction Enhancement Solution was then added dropwise, and the sections were rinsed repeatedly with 1× PBS after 30 min.

[0361] (3) Secondary antibody incubation: The secondary antibody was diluted 1:1000 in 1×PBS and added dropwise to the sections. The sections were kept in the dark at room temperature for 1-2 hours and then rinsed with 1×PBS for 5 minutes.

[0362] (4) Color development: Incubate the sections with DAB color development solution until the color turns brown, then immediately rinse with 1× PBS for 3 minutes, and repeat twice.

[0363] (5) Hematoxylin counterstaining: Immerse the sections in hematoxylin staining solution, stain for 5 minutes, then wash with water, add 1% hydrochloric acid for differentiation and dilute ammonia water for blueing.

[0364] (6) Dehydration and clearing: After washing the sections with tap water, dehydrate and clear them according to the following process: 75% alcohol, 2 min; 85% alcohol, 2 min; 95% alcohol, 2 min; anhydrous ethanol, 2 min; anhydrous ethanol, 2 min; 1 / 2 xylene, 5 min; xylene, 5 min; xylene, 5 min.

[0365] (7) Sealing: Add a drop of transparent nail polish on the slide, and slowly cover the slice with a cover slip from one side. After completion, observe under a microscope.

[0366] 3. Results and Analysis

[0367] (1) DEGs and their enriched signaling pathways in HUVEC treated with MSC-CM in a high-glucose environment

[0368] In order to further explore the molecular mechanism of hUC-MSCs on the angiogenesis of HUVEC in high glucose environment through paracrine, the present invention analyzed the transcriptome changes of HUVEC cells after high glucose (HG) and MSC-CM treatment by RNAseq. Figure 16 Figure -A shows an overview of differentially expressed genes (DEGs) in the three HUVEC cell normal treatment (Normal), high glucose treatment (HG), and hUC-MSCs culture supernatant treatment (MSC-CM) groups. The volcano plot results showed that based on p < 0.05 and |Log2 fold| > 1, 8323 and 198 DEGs were identified between the Normal and HG groups, and between the HG and MSC-CM groups, respectively. Figure 16 -B and Figure 16 -C.

[0369] GO pathway enrichment analysis results are as follows Figure 16 -D shows that in terms of cellular processes and cell signaling pathways, DEGs between the HG and MSC-CM groups were mainly enriched in muscle cytoskeleton, adhesion, phagocytosis, P53 signaling pathway, ferroptosis, PI3K-AKT signaling pathway, Hippo signaling pathway, TGF-β signaling pathway, extracellular matrix effect, etc., involving cellular functions such as HUVEC cell survival, proliferation, migration and angiogenesis. It is worth noting that the PI3K-AKT signaling pathway is enriched with the most DEGs, most of which are highly expressed genes, while the P53 signaling pathway that regulates cell apoptosis accounts for the majority of low-expression genes. KEGG pathway enrichment analysis also showed that the PI3K-AKT signaling pathway plays an important role in HUVEC angiogenesis, see for details. Figure 16 Therefore, this paper selected the PI3K-AKT signaling pathway for in-depth mechanism research.

[0370] The regulation of angiogenesis is a delicate and complex process involving the interaction of numerous signaling pathways and regulatory factors. RNA sequencing results have revealed the main role of the PI3K-AKT signaling pathway. This paper is based on the PI3K-AKT signaling pathway diagram of DEGs in the HG and MSC-CM groups (see Figure 17), further exploring the molecular mechanisms by which hUC-MSCs promote HUVEC angiogenesis in a high-glucose environment through paracrine secretion, revealed that PI3K-AKT and its downstream VEGF signaling pathways, MAPK signaling pathways, mTOR signaling pathways, and P53 signaling pathways play a crucial role in the survival and function of vascular endothelial cells. VEGF-A is a major regulator of vascular remodeling and permeability at the wound site. The VEGF / VEGFR signaling pathway is one of the most critical pro-angiogenic signaling pathways, promoting cell migration, proliferation, and angiogenesis through eNOS activation. The MAPK signaling pathway participates in the regulation of cell proliferation and migration through ERK activation. The mTOR signaling pathway plays an important role in cell growth and metabolism. P53 is a key regulator of cell apoptosis.

[0371] (2) hUC-MSCs paracrine regulation of HUVEC PI3K-AKT and its downstream signaling pathways

[0372] To further verify the results of RNA-seq analysis, the changes in the expression levels of related genes in the VEGF signaling pathway, MAPK signaling pathway, mTOR signaling pathway, and P53 signaling pathway mediated by PI3K-AKT in HUVEC cells were analyzed. Figure 18 As shown, the expression levels of p-AKT, p-eNOS, p-mTOR, and p-ERK / 2 in HUVEC cells exposed to high glucose were significantly lower than those in the normal group, indicating impairment of the VEGF, MAPK, and mTOR signaling pathways, consistent with the reduced cell viability, migration, and angiogenesis. The expression level of p53 was significantly higher in the normal group, indicating activated apoptosis and reduced cell viability, consistent with previous results. The expression levels of p-AKT, p-eNOS, p-mTOR, and p-ERK1 / 2 in the MSC-CM and combined treatment groups were significantly higher than those in the control and Met groups, while the expression level of p53 was significantly lower than that in the control and Met groups. This indicates that the culture supernatant of hUC-MSCs (MSC-CM) can significantly restore the VEGF, MAPK, and mTOR signaling pathways in HUVEC cells exposed to high glucose and inhibit the p53 signaling pathway. The synergistic effect of Met significantly enhances this effect, promoting the regulation of related pathways and thereby promoting HUVEC cell survival, proliferation, migration, and in vitro angiogenesis.

[0373] (3) hUC-MSCs paracrine activation of PI3K-AKT and its downstream signaling pathways in DFU wounds

[0374] To further explore the signaling pathway mechanism of hUC-MSCs paracrine ability on angiogenesis in DFU rat wounds, this paper conducted immunohistochemical experiments on the wounds of DFU rats 14 days after treatment to analyze the changes in the expression levels of related genes in the PI3K-AKT and its downstream VEGF signaling pathway, MAPK signaling pathway, mTOR signaling pathway, and P53 signaling pathway in DFU rats. The results of the immunohistochemical experiment and its quantitative results are shown in Figure 2. Figure 19 and Figure 20 As shown in the results, the positive rates of p-AKT, p-eNOS, p-mTOR, and p-ERK1 / 2 in DFU rats were significantly lower than those in the normal group, indicating that the VEGF signaling pathway, MAPK signaling pathway, and mTOR signaling pathway were impaired; while the positive rate of P53 was significantly higher than that in the normal group, indicating that the apoptosis pathway was activated. The positive expression of p-AKT, p-eNOS, p-mTOR, and p-ERK1 / 2 in the MSC-CM treatment group and the combined treatment group was significantly higher than that in the control group and the Met group, while the positive expression of P53 was significantly lower than that in the control group and the Met group. This indicates that hUC-MSCs significantly restored the VEGF signaling pathway, MAPK signaling pathway, and mTOR signaling pathway in DFU rats through paracrine function and inhibited the P53 signaling pathway. This is consistent with the previous results that hUC-MSCs promote angiogenesis and healing in DFU rats, and the synergistic effect of Met significantly enhanced this effect.

Claims

1. Application of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of drugs for the treatment of diabetic foot ulcers.

2. The use according to claim 1, characterized in that The human umbilical cord mesenchymal stem cells highly express CD73, CD90 and CD105, and lowly express CD34, CD45 and HLA-DR.

3. The use according to claim 1, characterized in that The diabetic foot ulcer is accompanied by weight loss, hyperglycemia, impaired glucose metabolism, insulin resistance, delayed wound healing, inflammation, impaired epithelialization, impaired angiogenesis and / or polydipsia and polyuria.

4. The use according to claim 1, characterized in that The drug is used to promote wound closure of diabetic foot ulcers; and / or the drug is used to promote re-epithelialization and collagen deposition in diabetic foot ulcer wounds; and / or the medicament is used to promote regeneration of skin appendages; And / or the drug is used to promote scarless wound healing and resist fibrotic scar formation; and / or the drug is used to promote wound healing and proliferation; And / or the drug is used to inhibit wound inflammation.

5. Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of angiogenesis promoters, cell proliferation promoters and / or cell migration promoters for the treatment of diabetic foot ulcers.

6. Application of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of hypoglycemic drugs.

7. Use of metformin in the preparation of a drug for inhibiting or repairing damage to human umbilical cord mesenchymal stem cells induced by a high-glucose environment.

8. The use according to claim 7, characterized in that The human umbilical cord mesenchymal stem cells are combined with metformin to restore the VEGF signaling pathway, MAPK signaling pathway, and mTOR signaling pathway in the body of DFU patients through paracrine function, and inhibit the P53 signaling pathway, thereby promoting wound angiogenesis and healing.

9. A pharmaceutical composition for treating diabetic foot ulcers and / or lowering blood sugar, characterized in that: The pharmaceutical composition comprises human umbilical cord mesenchymal stem cells and metformin.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is a combination drug; or the pharmaceutical composition is prepared in the form of a compound drug into any pharmaceutically acceptable dosage form.

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