Stem cell culture method capable of promoting wound healing and application of stem cell culture method

Abstract

The invention relates to the technical field of biological medicine, in particular to a stem cell culture method capable of promoting wound healing and application thereof. The stem cell culture method capable of promoting wound healing comprises the following steps: step 1, providing umbilical cord-derived mesenchymal stem cells; 2, luteolin and baicalin are added into the serum supplement culture medium to serve as culture medium additives, the concentration of the luteolin is 1-10 [mu] mol / L, and the concentration of the baicalin is 1-10 [mu] mol / L; 3, inoculating the mesenchymal stem cells into a culture medium containing an additive, and culturing for 48-96 hours under the culture conditions that the temperature is 34-40 DEG C, the CO2 content is 4-6% and the culture time is 48-96 hours; 4, the cultured mesenchymal stem cells are harvested, and the stem cells capable of promoting wound healing are obtained. According to the culture method, stem cells can be promoted to secrete multiple growth factors, secretion of PGE2 and VEGF-C growth factors is remarkably improved, the wound healing rate of a stem cell treatment group is increased to 96%, and the culture method has a remarkable effect on treating diabetic foot ulcer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a stem cell culture method and application thereof capable of promoting wound healing. Background Art

[0002] Diabetic foot ulcers are one of the most common serious complications of diabetes and a leading cause of amputation and even death. Currently, treatments for diabetic foot ulcers include debridement, anti-infection, revascularization, and wound repair. However, these traditional treatments are limited in effectiveness, especially for refractory ulcers, and clinical needs remain unmet.

[0003] In recent years, new therapies based on mesenchymal stem cells (MSCs) have become a research hotspot, especially umbilical cord-derived MSCs, which have shown good application prospects due to their high availability, high proliferation capacity, and low immunogenicity. The main mechanisms of action of stem cells in treating diabetic foot ulcers include:

[0004] (1) Promote angiogenesis: Stem cells promote endothelial cell proliferation, migration and vascular remodeling by secreting cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), thereby improving local microcirculation; (2) Immune regulation: Stem cells inhibit the release of inflammatory factors such as IL-6 and TNF-α by macrophages, while promoting the secretion of anti-inflammatory cytokines IL-10 and IL-12, thereby alleviating the inflammatory microenvironment and accelerating wound healing.

[0005] However, existing stem cell therapy technologies still have the following limitations: (1) Stem cells have different proliferation and viability in different culture systems. When stem cells are cultured in vitro using an unoptimized conventional culture system, their proliferation and viability are inferior to those of an optimized culture system, and this disadvantage affects the activity of stem cells in vivo. (2) The core mechanism of stem cell wound treatment is to indirectly promote angiogenesis, epithelial cell proliferation, and collagen deposition by secreting a variety of bioactive factors (such as vascular endothelial growth factor VEGF-C). However, the paracrine capacity of unoptimized stem cells is limited and cannot meet the needs of wound repair. (3) The existing method of optimizing stem cell culture systems by adding factors is costly, which increases the price of stem cell drugs and limits the clinical promotion of stem cell preparations.

[0006] Therefore, there is an urgent need to develop a new technical means that can improve the paracrine ability of stem cells in diabetic foot ulcers, enhance the vitality and functional activity of stem cells, and reduce its manufacturing cost to overcome the shortcomings of existing technologies. Summary of the Invention

[0007] In response to the deficiencies in the above-mentioned prior art, the present invention provides a stem cell culture method and application thereof for promoting wound healing. In order to improve the survival rate of mesenchymal stem cells in diabetic foot ulcers, this solution provides a mesenchymal stem cell culture medium additive. By adding luteolin and baicalin in proportion to the mesenchymal stem cell culture medium, MSCs with stronger and higher repair effects can be cultivated.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A stem cell culture method for promoting wound healing comprises adding luteolin and baicalin as culture medium additives to a serum supplemented culture medium.

[0010] Preferably, in the serum-supplemented culture medium, the concentration of luteolin is 1-10 μmol / L, and the concentration of baicalin is 1-10 μmol / L.

[0011] Preferably, the method comprises the following steps:

[0012] Step 1: Providing umbilical cord-derived mesenchymal stem cells;

[0013] Step 2: adding luteolin and baicalin as culture medium additives to the serum-supplemented culture medium;

[0014] Step 3: inoculating the mesenchymal stem cells into a culture medium containing additives for cultivation;

[0015] Step 4: harvesting the cultured mesenchymal stem cells to obtain stem cells that promote wound healing.

[0016] More preferably, the method comprises the following steps:

[0017] Step 1: Providing umbilical cord-derived mesenchymal stem cells;

[0018] Step 2: adding luteolin and baicalin as culture medium additives to the serum-supplemented culture medium, wherein the concentration of luteolin is 1-10 μmol / L, and the concentration of baicalin is 1-10 μmol / L;

[0019] Step 3: inoculating the mesenchymal stem cells into a culture medium containing additives and culturing them at 34-40°C, 4-6% CO2, and for 48-96 hours;

[0020] Step 4: harvesting the cultured mesenchymal stem cells to obtain stem cells that promote wound healing.

[0021] Preferably, the mesenchymal stem cells are mesenchymal stem cells derived from umbilical cord Wharton's jelly, whose surface markers CD90, CD29, CD166, CD105 and CD73 are positively expressed, and CD45, CD34, CD11b, CD19 and HLA-DR are negatively expressed.

[0022] Preferably, the serum-supplemented culture medium is α-MEM basal culture medium, and FBS is added at a volume fraction of 5%-25%.

[0023] Preferably, the mesenchymal stem cells in step 4 are stem cells at passage P4-P8.

[0024] Preferably, the factors secreted by the cultured mesenchymal stem cells include VEGF-C and PGE2.

[0025] Preferably, the cultured mesenchymal stem cells promote angiogenesis and lymphangiogenesis by activating VEGFR3-CaN-NFAT, EP3-PI3K / Akt, and Akt-eNOS signaling pathways.

[0026] Preferably, the culture medium additive further comprises isobutyric acid. The concentration of isobutyric acid in the serum-supplemented culture medium is 0.1-10 μmol / L; further, the concentration of isobutyric acid in the serum-supplemented culture medium is 0.5-5 μmol / L.

[0027] Preferably, the mesenchymal stem cells are used to treat diabetic foot ulcers.

[0028] The mesenchymal stem cells cultured by the method are used in the preparation of medicines for treating diabetic foot ulcers.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention provides a stem cell culture method and application for promoting wound healing. The addition of baicalin and luteolin during the stem cell culture process can provide a favorable microenvironment for stem cell growth and functional maintenance, and can increase the secretion levels of multiple growth factors in stem cells. The secretion of PGE2 in the cells was significantly increased, and the baicalin group also saw an increase. However, when luteolin and baicalin were added simultaneously, PGE2 secretion increased most significantly, reaching 2.13 times that of the basal group. The addition of baicalin can increase the secretion level of VEGF-C in umbilical cord mesenchymal stem cells. Similarly, when luteolin and baicalin were added simultaneously, VEGF-C secretion increased most significantly, reaching 1.88 times that of the basal group. This synergistic effect is due to the fact that luteolin enhances anti-inflammatory and angiogenic effects through the EP3-PI3K / Akt pathway, while baicalin specifically promotes lymphangiogenesis through the VEGFR3-CaN-NFAT pathway, forming a complementary dual regulatory network.

[0031] 2. Animal experiments of the present invention show that this culture method increases the wound healing rate of the stem cell treatment group to 96%, and the healing quality is significantly improved. The results show that the stem cells prepared by the present invention can effectively relieve chronic inflammation and promote wound healing; at the same time, it can reduce oxidative stress and fibrosis, prolong the survival time of stem cells and improve the quality of tissue repair.

[0032] 3. The luteolin and baicalin used in this invention are both naturally derived compounds, highly safe, and easily scalable. Compared to expensive growth factors such as VEGF and bFGF used in existing technologies, this significantly reduces production costs. A single injection of cultured stem cells can achieve a wound healing rate of over 96% within 15 days, providing an efficient and cost-effective solution for the standardized treatment of diabetic foot ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 Results of CCK-8 assay showing the effects of different culture medium additives on cell number and viability.

[0035] Figure 2 The results show the effects of different culture medium additives on cell secretion of PGE2.

[0036] Figure 3The results show the effects of different culture medium additives on cell secretion of VEGF-C.

[0037] Figure 4 The results of the wound healing rate after administering stem cells cultured with different culture medium additives to mouse skin defect model animals.

[0038] Figure 5 Comparative photos of skin defect wound healing at different time points after administration of stem cells cultured with different culture medium additives to mice.

[0039] Figure 6 The figure shows the pathological scoring of the skin defect wound tissue of mice 15 days after administration of stem cells cultured with different culture medium additives. DETAILED DESCRIPTION

[0040] The above content of the present invention will be further described in detail below in conjunction with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments.

[0041] Some of the raw materials described in this application are commercially available. Other raw materials not described are commercially available:

[0042] MEM Alpha (1X) Minimum Essential Medium was purchased from Thermo Fisher Scientific, catalog number: 12571063;

[0043] TrypLE™ Express enzyme (1X) was purchased from Thermo Fisher Scientific, catalog number: 12604013;

[0044] FOETAL BOVINESERUM was purchased from HyClone, product number: SV30208.02;

[0045] Trypan Blue Stain (0.4%) was purchased from Thermo Fisher Scientific, catalog number: 15250061;

[0046] CryoPur-DMSO was purchased from Origen, catalog number: CP-70;

[0047] Sodium chloride injection was purchased from Huaren Pharmaceutical Rizhao Co., Ltd., product number: National Medicine Standard H20023146;

[0048] CCK-8 kit was purchased from Shanghai Biotech Biotechnology Co., Ltd., catalog number: C0043;

[0049] Human VEGF-C Immunoassay Quantikine® ELISA was purchased from R&D Systems, catalog number: DVEC00;

[0050] Prostaglandin E2 Assay was purchased from R&D Systems, catalog number: KGE004B;

[0051] Luteolin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: B110209;

[0052] Baicalin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: L107329;

[0053] Isodanin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: I157676.

[0054] A stem cell culture method for promoting wound healing:

[0055] In the present invention, the stem cell culture method for promoting wound healing comprises the following steps:

[0056] 1. Cell Source

[0057] 1.1 Primary cell acquisition and culture

[0058] Aseptically collect fresh umbilical cord tissue after cesarean section. The collection length range is 5 cm-15 cm, preferably 10 cm. Rinse the umbilical cord tissue with sodium chloride injection to remove residual blood. Cut the umbilical cord into small segments of 1 cm-5 cm in length, preferably 2 cm-3 cm. Rinse again with sodium chloride injection. Cut the umbilical cord longitudinally, remove the umbilical vein and umbilical artery, peel off the Wharton's jelly tissue, and cut the Wharton's jelly tissue into 0.5 mm pieces. 3 -2 mm 3 Small tissue pieces, preferably 1 mm 3 Evenly seed the tissue pieces into a cell culture flask and add α-MEM medium containing 5%-20% fetal bovine serum (FBS), preferably 10% FBS. Place the flask in an incubator at 35-38°C and 4%-6% CO2, preferably 37°C and 5% CO2. Observe cell growth daily. When the cell confluence reaches 70-95%, preferably 80-90%, digest and passage the cells using recombinant trypsin for 1-10 minutes, preferably 3-5 minutes.

[0059] 1.2 Culture and cryopreservation of umbilical cord mesenchymal stem cells

[0060] After the primary cells are digested, they are inoculated into culture flasks and cultured for two generations. When the cell confluence reaches 90%, recombinant trypsin is used for digestion and passage. The digested cells are centrifuged at 800-1200 rpm for 3-10 min, preferably at 1000 rpm for 5 min, and the supernatant is discarded. The cells are resuspended in freezing solution, which consists of 60%-80% α-MEM, 5%-25% FBS, 5%-25% dimethyl sulfoxide (DMSO), preferably 70% α-MEM, 20% FBS, 10% DMSO. The cell suspension concentration is 1×10 6 cells / mL to 1×10 7 cells / mL, preferably 5×10 6 cells / mL. Aliquot into cryovials, with a volume of 0.5 mL-2 mL per tube, preferably 1 mL. Place the cryovials in a pre-chilled, programmed cooling box filled with isopropanol and cool to -80°C. The next day, remove the cryovials and transfer them to liquid nitrogen for storage. These are the P2 seed cells.

[0061] 1.3 Surface marker detection

[0062] Take the cells before freezing and prepare a concentration of 1×10 5 cells / mL-1×10 7 cells / mL, preferably 1×10 6 Cell surface markers were detected by flow cytometry. Positive markers included CD90, CD29, CD166, CD105, and CD73, while negative markers included CD45, CD34, CD11b, CD19, and HLA-DR. The concentration of fluorescently labeled antibodies was 0.1 μg / mL–10 μg / mL, preferably 1 μg / mL, and the incubation time was 15–60 min, preferably 30 min. The test results must meet the requirements of a positive marker expression rate ≥95% and a total negative marker expression rate ≤2%.

[0063] 1.4 Culture and Preparation of Umbilical Cord Mesenchymal Stem Cells

[0064] Remove the cryovial containing the frozen P2 cells from liquid nitrogen and thaw in a 35-40°C water bath for 1-3 minutes, preferably 37°C for 2 minutes. Transfer the cell suspension to a centrifuge tube containing prewarmed α-MEM basal medium and centrifuge at 800-1200 rpm for 3-10 minutes, preferably 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in prewarmed α-MEM basal medium. Count the cells using trypan blue staining.

[0065] Inoculate the cell suspension into a T25-T225 culture flask or cell factory at a density of 6,000-15,000 cells / cm², preferably 7,000 cells / cm². Add α-MEM medium (preferably 10% FBS) supplemented with 5%-20% (v / v) FBS, and incubate in an incubator at 35-38°C and 4-6% CO2 for 48-96 hours, preferably 37°C and 5% CO2 for 72 hours. This is passage 3 cells. Subculture to passage 4 using the same procedure. Observe cell growth daily and subculture when the cell confluence reaches 80%-95%, preferably 85%-95%.

[0066] During passaging, discard the culture supernatant, wash with DPBS 1-3 times, add 1-100 mL of recombinant trypsin, and digest for 1-10 minutes, preferably 3-5 minutes. Immediately add 1-3 volumes of DPBS to terminate the digestion. Gently pipette the culture surface to collect the cell suspension and centrifuge at 800-1200 rpm for 3-10 minutes, preferably 1000 rpm for 5 minutes. Discard the supernatant to harvest the P5 cells.

[0067] Preparation: Observe cell growth every day. When the cell confluence reaches 85%-95%, digest and harvest the cells (same method as above), centrifuge and discard the supernatant, resuspend in sodium chloride injection, and adjust the final concentration to 1×10 6 -1×10 7 cells / mL, preferably 5×10 6 cells / mL for subsequent preparations.

[0068] The present invention incorporates baicalin and luteolin into the culture medium. Baicalin, a flavonoid extracted from plants such as Scutellaria baicalensis, possesses various biological activities, including anti-inflammatory and antibacterial properties. As a component of the stem cell culture medium, it provides a favorable microenvironment for stem cell growth and functional maintenance, enhancing the secretion of multiple growth factors. Regarding angiogenesis and lymphangiogenesis, VEGF-C-VEGFR3 signaling promotes nitric oxide (NO) release through the Akt / eNOS pathway. NO, a potent vasodilator, increases local blood perfusion and activates MMP-2 / 9-mediated extracellular matrix remodeling, thereby promoting endothelial cell migration and angiogenesis. The VEGFR3-CaN-NFAT pathway specifically induces lymphatic endothelial cell proliferation and lymphangiogenesis, accelerating interstitial fluid return and alleviating ulcer edema. PGE2 further enhances VEGF / VEGFR2 signaling through the EP3-PI3K / Akt pathway, synergizing with VEGF-C to promote angiogenesis. Experiments have shown that the combined use of PGE2 and VEGF-C can increase the capillary density of diabetic full-thickness skin defect model mice by about 70% and the lymphatic vessel density by about 50%, significantly improving the efficiency of microcirculation reconstruction.

[0069] In terms of inflammation and immune regulation, PGE2 inhibits NF-κB nuclear translocation through the EP3 receptor, reducing the release of proinflammatory cytokines such as TNF-α and IL-6. It also induces macrophage polarization toward the anti-inflammatory M2 phenotype, promoting the secretion of IL-10 and TGF-β, thereby significantly alleviating the local inflammatory response in the ulcer. VEGF-C, by promoting lymphangiogenesis, increases the number and transport efficiency of "channels" between the inflammatory site and the draining lymph nodes. Inflammatory cells such as neutrophils and macrophages can be transported to the draining lymph nodes through the lymphatic vessels and subsequently cleared or "inactivated," thereby reducing inflammatory cell infiltration at the inflammatory site and further shortening the inflammatory phase. After MSCs treatment, serum levels of TNF-α, IL-6, and hs-CRP were significantly reduced, and the decrease in these inflammatory markers was negatively correlated with the rate of wound healing, highlighting the key role of inflammatory regulation in the repair of diabetic foot ulcers.

[0070] Isodan-emodin reduces the release of TNF-α and IL-6 inflammatory factors by inhibiting the NF-κB and MAPK pathways, thereby improving the inflammatory microenvironment of diabetic ulcers; at the same time, it upregulates the expression of VEGF and Ang-1, promotes endothelial cell migration and capillary formation, and reduces scar formation by inhibiting the TGF-β / Smad pathway.

[0071] Isodan-emodin, baicalin, and luteolin exert significant effects in stem cell culture through complementary signaling pathways and synergistic functions. Baicalin primarily activates the VEGFR3-CaN-NFAT and Akt-eNOS pathways to promote lymphangiogenesis, while luteolin enhances PGE2 secretion via EP3-PI3K / Akt to inhibit inflammation and synergistically promote angiogenesis. While isodan-emodin supplements the Ang-1 pathway to stabilize new blood vessels and alleviate oxidative stress. The combination of these three compounds forms multiple signaling pathways, significantly improving the efficiency of microcirculatory reconstruction and synergistically regulating inflammation and anti-oxidation to further optimize the stem cell therapeutic microenvironment.

[0072] Example 1

[0073] A method for culturing stem cells that promotes wound healing, comprising the following steps:

[0074] 1. Cell Source

[0075] 1.1 Primary cell acquisition and culture

[0076] Fresh umbilical cord tissue was collected aseptically after cesarean section. The collection length range was 10 cm. The umbilical cord tissue was rinsed with sodium chloride injection to remove residual blood. The umbilical cord was cut into small segments of 2.5 cm in length. After rinsing with sodium chloride injection again, the umbilical cord was cut longitudinally, the umbilical vein and umbilical artery were removed, and the Wharton's jelly tissue was peeled off. The Wharton's jelly tissue was cut into 1 mm pieces. 3 Small tissue fragments were evenly inoculated into cell culture flasks. α-MEM medium containing 10% fetal bovine serum (FBS) was added and the flasks were placed in a 37°C, 5% CO2 incubator for static culture. Cell growth was monitored daily. When the cell confluence reached 85%, cells were digested and passaged using recombinant trypsin for 4 minutes.

[0077] 1.2 Culture and cryopreservation of umbilical cord mesenchymal stem cells

[0078] After digestion, the primary cells were inoculated into culture flasks and cultured for two generations. When the cell confluency reached 90%, they were digested and passaged using recombinant trypsin. The digested cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. The cells were resuspended in freezing solution composed of 70% α-MEM, 20% FBS, and 10% DMSO. The cell suspension concentration was 5×10 6 cells / mL, and dispense into cryovials with a volume of 1 mL per tube. Place the cryovials in a pre-cooled programmed cooling box filled with isopropanol and cool to -80°C. On the second day, remove the cell cryovials and transfer them to liquid nitrogen for storage. This is the P2 seed cell.

[0079] 1.3 Surface marker detection: Take the cells before freezing and prepare a concentration of 1×10 6 Cell surface markers were measured using flow cytometry in a cell suspension of 100 cells / mL. Positive markers included CD90, CD29, CD166, CD105, and CD73, while negative markers included CD45, CD34, CD11b, CD19, and HLA-DR. The concentration of fluorescently labeled antibodies was 0.1 μg / mL to 10 μg / mL, preferably 1 μg / mL, and the incubation time was 30 minutes. The test results must meet the requirements of positive marker expression ≥95% and total negative marker expression ≤2%.

[0080] 1.4 Culture and Preparation of Umbilical Cord Mesenchymal Stem Cells

[0081] Take the frozen P2 cells out of the cryovial from liquid nitrogen and thaw them in a 37°C water bath for 2 min. Transfer the cell suspension into a centrifuge tube containing preheated α-MEM basal medium and centrifuge at 1000 rpm for 5 min. Discard the supernatant and resuspend the cells in preheated α-MEM basal medium. Count the cells using trypan blue staining.

[0082] According to the counting results, the cell suspension was inoculated into T225 culture flasks or cell factories with an inoculation density of 7000 cells / cm 2 , add to Medium A, consisting of 10 mL FBS + 90 mL α-MEM basal medium; culture in a 37°C, 5% CO2 incubator for 72 hours. These are the P3 seed cells. Observe cell growth daily and passage when cell confluence reaches 85%. Repeat the same procedure to culture to P4. Cryopreserve P4 cells according to the P2 freezing procedure.

[0083] During subculturing, discard the culture supernatant, wash three times with DPBS, and digest with recombinant trypsin for 4 minutes. Immediately add two volumes of DPBS to terminate digestion. Gently pipette the culture surface to collect the cell suspension, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to harvest P5 cells.

[0084] Preparation: When the cell confluence reaches 85%, digest and harvest the cells (same method as above), centrifuge and discard the supernatant, resuspend in sodium chloride injection, and adjust the final concentration to 5×10 6 cells / mL for subsequent experiments or preparations.

[0085] Example 2

[0086] The method is basically the same as Example 1, with the main differences being: 1.4 The culture medium for umbilical cord mesenchymal stem cells is different: the composition of the culture medium B is 10 mL FBS + 90 mL α-MEM basal medium + 0.715 mL luteolin solution (437 μmol / L).

[0087] The luteolin solution was prepared by dissolving 25 mg of luteolin in 0.2 mL of DMSO to obtain a 437 μmol / L luteolin solution.

[0088] Example 3

[0089] The method is basically the same as Example 1, with the main differences being: 1.4 The culture medium for umbilical cord mesenchymal stem cells is different: the composition of the culture medium C is 10 mL FBS + 90 mL α-MEM basal medium + 1.116 mL baicalin solution (280 μmol / L).

[0090] The baicalin solution was prepared by dissolving 25 mg of baicalin in 0.2 mL of DMSO to obtain a 280 μmol / L baicalin solution.

[0091] Example 4

[0092] The method is basically the same as Example 1, except that: 1.4 The culture medium for umbilical cord mesenchymal stem cells is different: the composition of the culture medium D is 10 mL FBS + 90 mL α-MEM basal medium + 0.358 mL luteolin solution (437 μmol / L) + 0.558 mL baicalin solution (280 μmol / L).

[0093] The preparation of the luteolin solution is the same as that in Example 2.

[0094] The preparation of the baicalin solution is the same as that in Example 3.

[0095] Example 5

[0096] The results are basically the same as those in Example 1, except that: 1.4 The culture medium for umbilical cord mesenchymal stem cells is different: the composition of the culture medium F is 10 mL FBS + 90 mL α-MEM basal medium + 0.238 mL luteolin solution (437 μmol / L) + 0.372 mL baicalin solution (280 μmol / L) + 0.359 mL isobutyric acid solution (290 μmol / L).

[0097] The preparation of the luteolin solution is the same as that in Example 2.

[0098] The preparation of the baicalin solution is the same as that in Example 3.

[0099] The isodanin solution was prepared by dissolving 15 mg of isodanin in 0.2 mL of DMSO to obtain a 290 μmol / L isodanin solution.

[0100] Comparative Example 1

[0101] The method is basically the same as Example 1, with the main differences being: 1.4 The culture medium for umbilical cord mesenchymal stem cells is different: the composition of the culture medium E is 10 mL FBS + 90 mL α-MEM basal medium + 1.078 mL isobutyric acid solution (290 μmol / L).

[0102] The preparation of the isodanin solution is the same as that in Example 5.

[0103] Test Case

[0104] 1. CCK-8 assay to detect the effects of different culture medium additives on cell viability

[0105] Experimental Procedure: Cell Recovery and Preparation: Preheat α-MEM basal medium to 37°C and place in a centrifuge tube for later use. To recover P4 umbilical cord mesenchymal stem cells, transfer the cell suspension to a centrifuge tube containing 9 mL of preheated α-MEM basal medium and mix thoroughly.

[0106] Preparation of cell suspension: The mixed cell suspension was divided equally into 6 centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and 1 mL of the culture medium prepared in Examples 1-5 and Comparative Example 1 was added to resuspend the cells. After thorough mixing, 20 μL of the cell suspension was added to 20 μL of 0.2% trypan blue staining solution and mixed, and viable cells were counted.

[0107] 96-well plate inoculation: Based on the cell count results, the concentration was adjusted to 40,000 cells / mL with the corresponding culture medium. 100 μL of DPBS was added to each well of the 96-well plate. 100 μL of cell suspension was placed in the 96-well plate and incubated at 37°C, 5% CO2 for 24 h. 10 μL of CCK-8 reagent was then added to each well and incubated for another 2 h. The 96-well plate was removed and the absorbance was measured at 450 nm using a microplate reader. The corresponding cell number, viability, and cell proliferation rate were calculated based on the absorbance. The results are shown in Table 1. Figure 1 and Table 1.

[0108] The absorbance value was measured at 450 nm using a microplate reader. The proliferation difference rate was calculated by subtracting the absorbance value of the blank control group from the absorbance value of each sample group. The proliferation difference rate = the absorbance value measured at 450 nm of each example or comparative example / the absorbance value measured at 450 nm of Example 1 × 100%.

[0109] Table 1 Cell proliferation difference rate

[0110]

[0111] As can be seen from the table above, the stem cells prepared by the present invention exhibited significant differences in cell proliferation rates in different culture media. The addition of either luteolin or baicalin alone significantly enhanced cell proliferation activity. Luteolin and baicalin are the core synergistic ingredients. When used in combination, luteolin's PI3K / Akt activation and baicalin's VEGFR3-NFAT signaling form cross-regulation, promoting endothelial cell proliferation (accelerating cell cycle progression) and migration (enhancing cytoskeleton remodeling) by activating downstream PI3K / Akt and MAPK (ERK1 / 2) signaling pathways. These dual pathways synergistically promote cell cycle protein expression, and dual signaling pathways synergistically activate cell growth. Simultaneously, the paracrine effects of PGE2 and VEGF-C further optimize the microenvironment, resulting in the highest differential proliferation rate. The proliferation promoting effect of isodanin added alone is weak. Isodanin is an auxiliary synergistic ingredient that further optimizes the proliferation effect by improving the microenvironment. When isodanin is used in combination with luteolin and baicalin, a multi-target network of anti-inflammatory (NF-κB inhibition), pro-proliferation (PI3K / Akt), and angiogenesis (VEGFR3 / Ang-1) is formed, which further reduces the cell apoptosis rate and increases the proliferation difference rate to 151.6%. This shows that isodanin further optimizes the proliferation microenvironment of stem cells, forms a triple synergistic effect with luteolin and baicalin, and has the largest proliferation difference rate.

[0112] 2. ELISA detection of TNF-α and VEGF secretion levels

[0113] Cell preparation: Preheat α-MEM basal medium to 37°C and place in a centrifuge tube for later use. To resuscitate P4 umbilical cord mesenchymal stem cells, transfer the cell cryopreservation solution to a centrifuge tube containing preheated culture medium and mix thoroughly.

[0114] Cell inoculation and culture: The mixed cell suspension was divided equally into 6 centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended with 1 mL of the culture medium prepared in Examples 1-5 and Comparative Example 1, respectively. After thorough mixing, 20 μL of the cell suspension was added to 20 μL of 0.2% trypan blue staining solution, mixed thoroughly, and viable cells were counted. According to the cell counting results, the cells in different groups were divided into 7000 cells / cm 2The cells were inoculated into T225 culture flasks, and the culture medium prepared in Examples 1-5 and Comparative Example 1 was added. The cells were cultured in a 37°C, 5% CO2 incubator for 72 h. When the cell confluence reached 90%, the cell supernatant was collected to detect the secretion levels of PGE2 and VEGF-C.

[0115] 2.1. PGE2 Assay: Prepare the required amounts of reagents in advance and set up nonspecific binding wells, blank control wells, standards, and test samples. Add 200 μL of RD5-56 diluent to the nonspecific binding wells (NSB); add 150 μL of RD5-56 diluent to the blank control well (B0); and add 150 μL of standard to the sample wells. Add 50 μL of Primary Antibody Solution to each well except the NSB well. Mix well five times with a dispenser. Seal the plate with a sealing film and incubate at room temperature with shaking at 550 rpm for 2 hours. Add 50 μL of PGE2 Conjugate directly to each well. Mix well five times with a dispenser. Seal the plate with a sealing film and incubate at room temperature with shaking at 500 rpm for 2.5 hours. Discard any liquid from the wells and add 400 μL of Wash Buffer (1×) to each well. Let stand for 90 seconds. Repeat the wash four times and pat dry. Add 200 μL of TMB substrate colorimetric solution to each well, seal the plate with a sealing film, and incubate at room temperature at 500 rpm in the dark for 30 min. Add 100 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the reaction. Read the absorbance at 450 / 540 nm on a microplate reader within 30 min. Figure 2 .

[0116] 2.2 VEGF-C Assay: Prepare the required amounts of reagents in advance, set up the standard, positive control, blank control, and test sample. Pipette 50 μL of the diluted standard, positive control, blank control, and test sample into the corresponding wells. Then, add 100 μL / well of Assay Diluent RD1W diluent to each well. Seal the plate with a sealing film and incubate at room temperature at 500 rpm for 2 h. After incubation, discard the liquid from the wells and add 400 μL of wash solution (1×) to each well. Repeat the wash four times and pat dry any remaining liquid. Add 200 μL of Human VEGF-C Conjugate HRP to each well and incubate at room temperature at 500 rpm for 2 h. After washing, add 200 μL of TMB substrate colorimetric solution to each well. Seal the plate with a sealing film and incubate at room temperature in the dark for 30 min. Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to terminate the reaction. Read the absorbance at 450 / 540 nm on a microplate reader within 30 min. Figure 3 .

[0117] 3. Animal experiments

[0118] To establish a diabetic full-thickness skin defect model in mice, 70 8-week-old BKS-db male mice were purchased. All mice were acclimated for 1 week and enrolled if their fasting blood glucose level was ≥11.1 mmol / L. Back hair was removed with a depilatory cream, and the back skin was disinfected with 75% alcohol and iodine. A 10 mm diameter circular full-thickness skin defect wound was created on the back of the mice. The wound was cared for on the day of modeling. Mice with successful modeling were randomly divided into 7 groups and administered the following medications according to Table 1:

[0119] Table 1. Dosage groups of mice

[0120]

[0121] Cell culture: Preheat the α-MEM basal culture medium to 37°C for use, and place it in a centrifuge tube for use; resuscitate the P4 generation umbilical cord mesenchymal stem cells, transfer the cell freezing solution to a centrifuge tube containing preheated culture medium, and mix thoroughly. Divide the mixed cell suspension into 6 centrifuge tubes, centrifuge at a centrifugal speed of 1000 rpm for 5 min, discard the supernatant, and resuspend the cells with 1 mL of the culture medium prepared in Examples 1-5 and Comparative Example 1, respectively. After thorough mixing, take 100 μL of the cell suspension, dilute it 10 times, and mix it thoroughly with 20 μL of 0.2% trypan blue staining solution to count the living cells. According to the cell counting results, the cells in different groups were counted at 7000 cells / cm 2 The cells were inoculated into a four-layer cell factory, and the culture medium prepared in Examples 1-5 and Comparative Example 1 was added. The cells were cultured in a 37°C, 5% CO2 incubator for 72 h. When the cell confluence reached 90%, the cells were digested and harvested, the supernatant was discarded, and the cells were resuspended in sodium chloride injection to adjust the concentration to 5×10 6 cells / mL.

[0122] Dosing regimen for mice: Each mouse in the treatment group received an injection of cell suspension;

[0123] Injection site: 4 points around the wound surface, up, down, left, and right;

[0124] Injection volume: 50 μL per point;

[0125] Total injection volume: 200 μL / mouse.

[0126] On days 0, 3, 7, 12, and 15, a standard ruler was placed during each observation to calculate the wound healing status and take photos of the wound to record the healing status. The results are as follows: Figure 4 and Figure 5 .

[0127] 4. Analysis of experimental results:

[0128] Figure 1 Compared with Group A, both the addition of luteolin and baicalin alone and the simultaneous addition of luteolin and baicalin increased cell number and viability (P < 0.05 at most time points), significantly enhancing the viability of umbilical cord mesenchymal stem cells. In Group D, the simultaneous addition of luteolin and baicalin resulted in the most significant increase in cell viability (P < 0.01), indicating a synergistic effect between the two.

[0129] Figure 2 In the experiment, the addition of luteolin significantly promoted the secretion of PGE2, which was 1.84 times that of Group A. Baicalin also had a promoting effect, which was 1.14 times that of Group A. When luteolin and baicalin were added at the same time, the secretion of PGE2 was the highest, which was 2.13 times that of Group A. The literature shows that PGE2 is an autocrine / paracrine signaling factor that promotes proliferation and survival, and umbilical cord mesenchymal stem cells themselves can produce and respond to it. PGE2 exerts its effect by binding to G protein-coupled receptors on its cell surface. The applicant speculates that the addition of baicalin and luteolin may act on a common upstream regulatory point, which can both activate the pro-proliferation pathway and induce the synthesis of PGE2.

[0130] Figure 3 In the study, the addition of baicalin significantly increased VEGF-C secretion, which was 1.57 times that of Group A. Luteolin had a weaker effect, at 1.19 times that of Group A. The optimal effect was achieved when both luteolin and baicalin were added, at 1.88 times that of Group A. VEGF-C is a highly specific vascular endothelial growth factor that promotes increased vascular permeability, endothelial cell migration, proliferation, and angiogenesis. Umbilical cord mesenchymal stem cells can produce and respond to it. After binding to VEGFR-2 / VEGFR-3 receptors on the surface of vascular endothelial cells, VEGF-C activates downstream signaling pathways such as PI3K / Akt and MAPK (ERK1 / 2), promoting endothelial cell proliferation (accelerating cell cycle progression) and migration (enhancing cytoskeleton remodeling ability), and inducing endothelial cells to form tubular structures (a core step in angiogenesis). In addition, VEGF-C can work synergistically with other pro-angiogenic factors (such as VEGF-A and bFGF) secreted by umbilical cord mesenchymal stem cells to amplify the pro-angiogenic effect through "signal superposition" and accelerate the recovery of blood supply to ischemic or damaged tissues.

[0131] Figure 4In the experiment, the stem cell treatment group promoted wound healing compared with the blank control group at both D12 and D15. Compared with the culture group without additives (Group A), the addition of baicalin or luteolin alone showed higher healing rates at both D12 and D15. The combined addition of baicalin and luteolin (Group D) showed the highest wound healing rate at all test time points, with the healing rate at D12 reaching 82% (the healing rate ranged from 32% to 76% when no additives were added or only one additive was added), and the healing rate at D15 was as high as 96% (the healing rate ranged from 36% to 82% when no additives were added or only one additive was added).

[0132] Figure 5 The blank group showed delayed wound healing, with obvious inflammation and edge contraction; group A showed only partial improvement, while group D showed faster wound re-epithelialization when luteolin and baicalin were added at the same time. Figure 4 The data were consistent, showing that the combined addition of the two additives enhanced the activity of umbilical cord mesenchymal stem cells in mice and promoted faster wound healing.

[0133] Figure 6 On day 15, tissue samples were collected from the wounds of animals in groups B, C, and D, and stained with hematoxylin and eosin (HE). Granulation and inflammatory cell infiltration were observed under a microscope. The group receiving both luteolin and baicalin (group D) showed increased granulation compared to the group receiving either luteolin alone (group B, P < 0.05) or baicalin alone (group C, P > 0.05). Inflammation levels at the wounds were significantly lower (P > 0.05) than in the groups receiving either luteolin alone. Stem cells cultured with both luteolin and baicalin may suppress local wound inflammation, modulate immunity to create an anti-inflammatory microenvironment, and promote angiogenesis to improve the blood supply microenvironment, ultimately enhancing the tissue repair capacity of umbilical cord mesenchymal stem cells through increased secretion of protease inhibitory gene (PGE2) and vascular endothelial growth factor (VEGF-C).

[0134] In summary, the addition of baicalin and luteolin can improve the cell viability and proliferation ability of umbilical cord mesenchymal stem cells, increase the secretion levels of VEGF-C and PGE2 of umbilical cord mesenchymal stem cells, activate the "PI3K / Akt / mTOR pathway" signaling pathway, thereby further enhancing the ability of umbilical cord mesenchymal stem cells to promote angiogenesis and inhibit inflammation, and significantly promote the healing effect of umbilical cord mesenchymal stem cells on diabetic foot ulcer wounds.

Claims

1. A method for culturing stem cells to promote wound healing, characterized in that: Luteolin and baicalin were added as medium additives in serum-supplemented medium.

2. The stem cell culture method for promoting wound healing according to claim 1, wherein: In the serum-supplemented culture medium, the concentration of luteolin is 1-10 μmol / L, and the concentration of baicalin is 1-10 μmol / L.

3. The stem cell culture method for promoting wound healing according to claim 2, wherein: The steps include: Step 1: Providing umbilical cord-derived mesenchymal stem cells; Step 2: adding luteolin and baicalin as culture medium additives to the serum-supplemented culture medium; Step 3: inoculating the mesenchymal stem cells into a culture medium containing additives for cultivation; Step 4: harvesting the cultured mesenchymal stem cells to obtain stem cells that promote wound healing.

4. The stem cell culture method for promoting wound healing according to claim 3, wherein: The steps include: Step 1: Providing umbilical cord-derived mesenchymal stem cells; Step 2: adding luteolin and baicalin as culture medium additives to the serum-supplemented culture medium, wherein the concentration of luteolin is 1-10 μmol / L, and the concentration of baicalin is 1-10 μmol / L; Step 3: inoculating the mesenchymal stem cells into a culture medium containing additives and culturing them at 34-40°C, 4-6% CO2, and for 48-96 hours; Step 4: harvesting the cultured mesenchymal stem cells to obtain stem cells that promote wound healing.

5. The stem cell culture method for promoting wound healing according to claim 3 or 4, wherein: The mesenchymal stem cells are derived from umbilical cord Wharton's jelly, and their surface markers CD90, CD29, CD166, CD105 and CD73 are positively expressed, and CD45, CD34, CD11b, CD19 and HLA-DR are negatively expressed.

6. The method for culturing stem cells for promoting wound healing according to any one of claims 1 to 4, wherein: The serum-supplemented culture medium is an α-MEM basal culture medium, and is supplemented with FBS having a volume fraction of 5%-25%.

7. The stem cell culture method for promoting wound healing according to claim 3 or 4, wherein: The mesenchymal stem cells in step 4 are stem cells at passage P4-P8.

8. The stem cell culture method for promoting wound healing according to claim 3 or 4, wherein: The factors secreted by the cultured mesenchymal stem cells include VEGF-C and PGE2.

9. The stem cell culture method for promoting wound healing according to claim 8, wherein: The cultured mesenchymal stem cells promote angiogenesis and lymphangiogenesis by activating VEGFR3-CaN-NFAT, EP3-PI3K / Akt, and Akt-eNOS signaling pathways.

10. Use of mesenchymal stem cells cultured according to the method of claim 9 in preparing a medicament for treating diabetic foot ulcers.

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