A stem cell culture method for promoting wound healing and its application

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a stem cell culture method for promoting wound healing and application thereof. The stem cell culture method for promoting wound healing comprises the following steps: step one, providing umbilical cord-derived mesenchymal stem cells; step two, adding luteolin and baicalin as culture medium additives in a serum-supplemented culture medium, wherein the concentration of luteolin is 1-10 micromoles / L, and the concentration of baicalin is 1-10 micromoles / L; step three, inoculating the mesenchymal stem cells into the culture medium containing the additives for culture, and the culture condition is 34-40 DEG C, 4-6% CO2, and the culture time is 48-96 hours; step four, harvesting the cultured mesenchymal stem cells to obtain stem cells for promoting wound healing. The culture method can promote the stem cells to secrete various growth factors, significantly improve the secretion of PGE2 and VEGF-C growth factors, and make the wound healing rate of the stem cell treatment group reach 96%, which has a significant effect on treating diabetic foot ulcers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a stem cell culture method for promoting wound healing and application thereof. BACKGROUND

[0002] Diabetic foot ulcer is one of the most common and severe complications of diabetes, and is also the main cause of amputation and even death of patients. At present, the treatment methods for diabetic foot ulcer include debridement, anti-infection, vascular reconstruction and wound repair, etc. However, the effect of traditional treatment methods is limited, especially for refractory ulcers, and the clinical needs have not been fully met.

[0003] In recent years, new therapies based on mesenchymal stem cells (MSCs) have become a research hotspot, especially umbilical cord-derived mesenchymal stem cells, which have shown good application prospects due to their strong availability, high proliferation capacity and low immunogenicity. The mechanism of stem cell therapy for diabetic foot ulcer mainly includes:

[0004] (1) Promoting angiogenesis: stem cells secrete cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), promote endothelial cell proliferation, migration and vascular remodeling, and improve local microcirculation; (2) Immune regulation: stem cells inhibit the release of inflammatory factors from macrophages, including IL-6, TNF-α, etc., while promoting the secretion of anti-inflammatory cytokines IL-10, IL-12, reducing the inflammatory microenvironment and accelerating wound healing.

[0005] However, the existing stem cell therapy technology still has the following limitations: (1) The proliferation capacity and viability of stem cells are different in different culture systems. The proliferation capacity and viability of stem cells cultured in vitro using conventional culture systems that have not been optimized are inferior to those in optimized culture systems, and this inferiority will affect the activity of stem cells in vivo; (2) The core mechanism of stem cell therapy for wounds is to indirectly promote angiogenesis, epithelial cell proliferation and collagen deposition by secreting various bioactive factors (such as vascular endothelial growth factor VEGF-C, etc.). However, the paracrine ability of unoptimized stem cells is limited, which is difficult to meet the needs of wound repair; (3) The existing method of optimizing stem cell culture system by adding factors and other means is costly, which raises the selling price of stem cell drugs and limits the clinical promotion of stem cell preparations.

[0006] Therefore, it is urgent to develop a new technical means that can improve the paracrine ability of stem cells at the site of diabetic foot ulcer, enhance the viability and functional activity of stem cells, and reduce the manufacturing cost, in order to overcome the shortcomings of the existing technology. SUMMARY

[0007] In view of the above problems in the prior art, the present application provides a stem cell culture method for promoting wound healing and application thereof, in order to improve the survival rate of mesenchymal stem cells at a diabetic foot ulcer, the present application provides a culture medium additive for mesenchymal stem cells, and the additive is added into a mesenchymal stem cell culture medium in a proportion of luteolin and baicalin, so that MSCs with stronger and higher repair effect can be cultured.

[0008] To solve the above technical problems, the technical scheme adopted by the present application is:

[0009] A stem cell culture method for promoting wound healing, wherein luteolin and baicalin are added into a serum-supplemented culture medium as a culture medium additive.

[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 one, providing umbilical cord-derived mesenchymal stem cells;

[0013] Step two, adding luteolin and baicalin into a serum-supplemented culture medium as a culture medium additive;

[0014] Step three, inoculating the mesenchymal stem cells into the culture medium containing the additive for culture;

[0015] Step four, harvesting the cultured mesenchymal stem cells to obtain stem cells with the function of promoting wound healing.

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

[0017] Step one, providing umbilical cord-derived mesenchymal stem cells;

[0018] Step two, adding luteolin and baicalin into a serum-supplemented culture medium as a culture medium additive, wherein the concentration of luteolin is 1-10 μmol / L, and the concentration of baicalin is 1-10 μmol / L;

[0019] Step three, inoculating the mesenchymal stem cells into the culture medium containing the additive for culture, and the culture conditions are 34-40℃ and 4-6% CO2, and the culture time is 48-96 h;

[0020] Step four, harvesting the cultured mesenchymal stem cells to obtain stem cells with the function of promoting wound healing.

[0021] Preferably, the mesenchymal stem cells are umbilical cord Wharton's jelly-derived mesenchymal stem cells, which are positive for surface markers CD90, CD29, CD166, CD105 and CD73, and negative for CD45, CD34, CD11b, CD19 and HLA-DR.

[0022] Preferably, the serum-supplemented medium is α-MEM basal medium supplemented with 5%-25% FBS.

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

[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, Akt-eNOS signaling pathways.

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

[0027] Preferably, the mesenchymal stem cells are used for treating diabetic foot ulcers.

[0028] The mesenchymal stem cells cultured by the method are used for preparing a medicament for treating diabetic foot ulcers.

[0029] The beneficial effects of the present application are as follows:

[0030] 1. The application provides a stem cell culture method for promoting wound healing and its application, which can provide a favorable microenvironment for the growth and function maintenance of stem cells after adding baicalin and luteolin during the stem cell culture process, and can improve the secretion level of various growth factors of stem cells. The secretion of PGE2 of the cells is significantly increased, and the baicalin group also increases, but the secretion of PGE2 is most obviously increased when baicalin and luteolin are added at the same time, and the secretion amount of PGE2 reaches 2.13 times of the basic group. The addition of baicalin can improve the secretion level of VEGF-C of umbilical cord mesenchymal stem cells, and when baicalin and luteolin are added at the same time, the secretion level of VEGF-C is most obviously improved, and the secretion amount of VEGF-C reaches 1.88 times of the basic group. This synergistic effect is due to the fact that luteolin enhances the anti-inflammatory and angiogenesis effect through the EP3-PI3K / Akt pathway, and baicalin specifically promotes lymphangiogenesis through the VEGFR3-CaN-NFAT pathway, and the two complement each other to form a double regulation network.

[0031] 2. Animal experiments of the application show that the culture method can improve the wound healing rate of the stem cell treatment group to 96%, and the healing quality is significantly improved, and the results show that the stem cells prepared by the application can effectively relieve chronic inflammation, promote wound healing; at the same time, 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 the application are natural source compounds, which are safe and easy to scale up. Compared with the expensive growth factors such as VEGF and bFGF in the prior art, the production cost is significantly reduced. Only a single injection of cultured stem cells can achieve a wound healing rate of more than 96% within 15 days, which provides an efficient and economical solution for the standardized treatment of diabetic foot ulcers. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0034] Figure 1 CCK-8 was used to detect the effect of different culture medium additives on cell number and viability.

[0035] Figure 2 The effect of different culture medium additives on cell secretion PGE2 was detected.

[0036] Figure 3The results of the influence of different medium additives on the secretion of VEGF-C by cells.

[0037] Figure 4 The results of the healing rate of the skin defect wound of the mouse administered with stem cells cultured in different medium additives.

[0038] Figure 5 The comparison of the skin defect wound healing of the mouse administered with stem cells cultured in different medium additives at different time points.

[0039] Figure 6 The pathological score of the skin defect wound of the mouse administered with stem cells cultured in different medium additives for 15 days. DETAILED DESCRIPTION

[0040] The above summary of the application will be further described in detail in combination with specific embodiments, but it should not be understood that the above summary of the application is limited to the following examples.

[0041] In this application, some raw materials are introduced, and other raw materials not introduced are commercially available:

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

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

[0044] FOETAL BOVINE SERUM purchased from HyClone, item number: SV30208.02;

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

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

[0047] Sodium Chloride Injection purchased from Huaren Pharmaceutical Rizhao Co., Ltd., item number: National Drug Standard Code H20023146;

[0048] CCK-8 kit purchased from Shanghai Blue Sky Bio-Technology Co., Ltd., item number: C0043;

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

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

[0051] Luteolin was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., item number: B110209;

[0052] Baicalin was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., item number: L107329;

[0053] Isorhapontigenin was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., item number: I157676.

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

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

[0056] 1. Cell source

[0057] 1.1 Primary cell acquisition and culture

[0058] Fresh umbilical cord tissue after cesarean section was collected aseptically, the length range was 5 cm-15 cm, preferably 10 cm, sodium chloride injection was used to flush the umbilical cord tissue to remove residual blood, the umbilical cord was cut into small pieces with a length of 1 cm-5 cm, preferably 2 cm-3 cm, 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 small tissue blocks with a size of 0.5 mm 3 -2 mm 3 , preferably 1 mm 3 , the tissue blocks were evenly inoculated in a cell culture bottle, and alpha-MEM culture medium containing 5%-20% fetal bovine serum (FBS) by volume, preferably 10% FBS, was added. The culture bottle was placed in a culture incubator with a temperature of 35-38 ℃ and a CO2 concentration of 4%-6%, preferably 37 ℃ and 5% CO2. The cell growth was observed daily, and when the cell confluence reached 70-95%, preferably 80-90%, the cells were passaged using recombinant trypsin digestion, and the digestion time was 1-10 min, preferably 3-5 min.

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

[0060] After the primary cells are digested and inoculated into culture bottles for continuous culture for 2 generations, when the cell confluence reaches 90%, the cells are passaged using recombinant trypsin. 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 using a cryopreservation solution, and the cryopreservation solution comprises 60%-80% α-MEM, 5%-25% FBS, and 5%-25% dimethyl sulfoxide (DMSO), preferably 70% α-MEM, 20% FBS, and 10% DMSO. The concentration of the cell suspension is 1×10 6 cells / mL to 1×10 7 cells / mL, preferably 5×10 6 cells / mL. The cells are aliquoted into cryopreservation tubes, with a volume of 0.5 mL-2 mL per tube, preferably 1 mL. The cryopreservation tubes are placed in a programmed cooling box containing isopropanol, cooled to -80°C, and then the cell cryopreservation tubes are removed on the second day and transferred to liquid nitrogen for storage, which is the P2 generation of seed cells.

[0061] 1.3 Surface marker detection

[0062] The cells before cryopreservation are taken, and a cell suspension with a concentration of 1×10 5 cells / mL-1×10 7 cells / mL is prepared, preferably 1×10 6 cells / mL. The cell surface markers are detected using a flow cytometer, and the positive markers detected include CD90, CD29, CD166, CD105, and CD73, and the negative markers include CD45, CD34, CD11b, CD19, and HLA-DR. During detection, the concentration of the fluorescently labeled antibody is 0.1 μg / mL-10 μg / mL, preferably 1 μg / mL, and the incubation time is 15 min-60 min, preferably 30 min. The detection results need to meet the requirements that the expression rate of the positive markers is ≥95% and the total expression rate of the negative markers is ≤2%.

[0063] 1.4 Culture and preparation of umbilical cord mesenchymal stem cells

[0064] The cryopreservation tube containing the P2 generation of cells is removed from liquid nitrogen, thawed in a 35-40 ℃ water bath for 1-3 min, preferably at 37 ℃ for 2 min. The cell suspension is transferred to a centrifuge tube containing preheated α-MEM basic medium, and centrifuged at 800-1200 rpm for 3-10 min, preferably at 1000 rpm for 5 min. The supernatant is discarded, and the cells are resuspended with preheated α-MEM basic medium, and counted using the trypan blue staining method.

[0065] The cell suspension is inoculated into T25-T225 culture flasks or cell factories at a seeding density of 6000-15,000 cells / cm2, preferably 7000 cells / cm2, and is cultured in different groups of α-MEM medium containing 5%-20% (v / v) FBS, preferably 10% FBS, in an incubator at 35-38 ℃ and a CO2 concentration of 4-6%, preferably at 37 ℃, 5% CO2, for 72 h, which is the P3 generation of cells, and the same operation is used for subculture to the P4 generation. The cell growth is observed every day, and the cells are subcultured when the cell confluence reaches 80%-95%, preferably 85%-95%.

[0066] At the time of subculture, the culture supernatant is discarded, and the cells are washed with DPBS for 1-3 times, 1-100 mL of recombinant trypsin is added, and the digestion time is 1-10 min, preferably 3-5 min, and 1-3 times the volume of DPBS is immediately added to terminate the digestion. The culture surface is gently blown, and the cell suspension is collected and centrifuged at 800-1200 rpm for 3-10 min, preferably at 1000 rpm for 5 min. The supernatant is discarded, and the P5 generation of cells is obtained.

[0067] Preparation of the preparation: The cell growth is observed every day, and when the cell confluence reaches 85%-95%, the cells are harvested by digestion (the method is the same as above), the supernatant is discarded after centrifugation, the cells are resuspended with sodium chloride injection, and the final concentration is adjusted to 1×10 6 -1×10 7 cells / mL, preferably 5×10 6 cells / mL, which is used for subsequent preparation.

[0068] The present application adds baicalin and luteolin in the culture medium, baicalin is a flavonoid compound extracted from plants such as scutellaria, has various biological activities, such as anti-inflammatory, antibacterial and other effects, in the present application as one of the components of stem cell culture medium, can provide a favorable microenvironment for the growth and function maintenance of stem cells, and can improve the secretion level of various growth factors of stem cells. In the aspect of blood vessel and lymphatic vessel neogenesis, VEGF-C-VEGFR3 signal promotes the release of nitric oxide (NO) through Akt / eNOS pathway, NO as a strong vasodilator can increase local blood perfusion, at the same time activates MMP-2 / 9 mediated extracellular matrix remodeling, and then promotes endothelial cell migration and blood vessel neogenesis. VEGFR3-CaN-NFAT pathway specifically induces lymphatic endothelial cell proliferation and lymphatic vessel formation, accelerates tissue fluid reflux and reduces ulcer edema. PGE2 further enhances VEGF / VEGFR2 signal through EP3-PI3K / Akt pathway, and synergistically promotes blood vessel neogenesis with VEGF-C. Experiments show that the combination 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 the aspect of inflammation and immune regulation, PGE2 inhibits NF-κB nuclear translocation through EP3 receptor, reduces the release of TNF-α, IL-6 and other pro-inflammatory factors, at the same time induces macrophages to polarize to anti-inflammatory M2 phenotype, promotes IL-10 and TGF-β secretion, thereby significantly alleviating the inflammatory response in the local ulcer. VEGF-C increases the number and transportation efficiency of "channels" between the inflammatory site and the draining lymph node by promoting lymphatic vessel neogenesis, and the inflammatory cells such as neutrophils and macrophages can be transported to the draining lymph node through the lymphatic vessels, and then be cleared or "inactivated", thereby reducing the inflammatory cell infiltration in the inflammatory site and further shortening the inflammation period. After MSCs treatment, the levels of TNF-α, IL-6 and hs-CRP in serum are significantly reduced, and the decrease of these inflammatory indicators is negatively correlated with the healing rate of the wound, highlighting the key role of inflammation regulation in the repair of diabetic foot ulcers.

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

[0071] Isorhapontigenin, baicalin and luteolin play a significant role in stem cell culture through complementary signal pathways and functional synergy. Baicalin mainly activates VEGFR3-CaN-NFAT and Akt-eNOS pathways to promote lymphangiogenesis, luteolin enhances PGE2 secretion through EP3-PI3K / Akt to inhibit inflammation and synergistically promote angiogenesis, while isorhapontigenin complements Ang-1 pathway to stabilize the newly formed blood vessels and reduce oxidative stress. The combination of the three forms a multi-signal pathway, significantly improves the efficiency of microcirculation reconstruction, and synergistically regulates inflammation and antioxidant to further optimize the therapeutic microenvironment of stem cells.

[0072] Example 1

[0073] A stem cell culture method with promoting wound healing, comprising the following steps:

[0074] 1. Cell source

[0075] 1.1 Primary cell acquisition and culture

[0076] Sterile collection of fresh umbilical cord tissue after cesarean section, the length range is 10 cm, use sodium chloride injection to flush the umbilical cord tissue to remove residual blood, cut the umbilical cord into small pieces with a length of 2.5 cm, rinse again with sodium chloride injection, then cut the umbilical cord longitudinally, remove the umbilical vein and umbilical artery, and peel off the Wharton's jelly tissue. Cut the Wharton's jelly tissue into small tissue blocks with a size of 1 mm 3 , evenly inoculate the tissue blocks into cell culture bottles, add α-MEM medium containing 10% fetal bovine serum (FBS) by volume fraction, and place the culture bottles in a CO2 incubator with a temperature of 37 ℃ and a concentration of 5%. Observe the cell growth every day, and when the cell confluence reaches 85%, use recombinant trypsin for digestion and passage, with a digestion time of 4 min.

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

[0078] After the primary cells are digested and inoculated into culture bottles for continuous culture for 2 generations, when the cell confluence reaches 90%, use recombinant trypsin for digestion and passage. Centrifuge the digested cells at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells with cryopreservation solution, which consists of 70% α-MEM, 20% FBS and 10% DMSO. The cell suspension concentration is 5×10 6 cells / mL, aliquot into cryopreservation tubes, each tube contains 1 mL, place the cryopreservation tubes into a pre-cooled program cooling box containing isopropanol, cool to -80 ℃, then on the second day, transfer the cell cryopreservation tubes to liquid nitrogen for storage, which is P2 generation seed cells.

[0079] 1.3 Surface marker detection: Take the cells before freezing, prepare a cell suspension with a concentration of 1 x 10 6 cells / mL, and detect the cell surface markers by flow cytometry. The positive markers include CD90, CD29, CD166, CD105, and CD73, and the negative markers include CD45, CD34, CD11b, CD19, and HLA-DR. During detection, the concentration of fluorescently labeled antibodies is 0.1 μg / mL-10 μg / mL, preferably 1 μg / mL, and the incubation time is 30 min. The detection results need to meet the following conditions: the expression rate of positive markers is ≥95%, and the total expression rate of negative markers is ≤2%.

[0080] 1.4 Culture and preparation of umbilical cord mesenchymal stem cells

[0081] Take the frozen P2 generation cells from the cryopreservation tube in liquid nitrogen, thaw them in a 37 ℃ water bath for 2 min, transfer the cell suspension to a centrifuge tube containing preheated α-MEM basic medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with preheated α-MEM basic medium, and count them using the trypan blue staining method.

[0082] According to the counting results, inoculate the cell suspension into a T225 culture flask or a cell factory with a seeding density of 7000 cells / cm 2 , add it to medium A composed of 10 mL FBS + 90 mL α-MEM basic medium, and place it in a 37 ℃ incubator with a CO2 concentration of 5% for 72 h, which is the P3 generation seed cell. Observe the cell growth every day, and subculture when the cell confluence reaches 85%. Subculture to the P4 generation by the same operation. Freeze the P4 generation cells according to the P2 generation freezing procedure.

[0083] During subculture, discard the culture supernatant, wash it with DPBS for 3 times, add recombinant trypsin for 4 min, immediately add twice the volume of DPBS to terminate the digestion. Gently blow the culture surface, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and obtain the P5 generation cells.

[0084] Preparation: When the cell confluence reaches 85%, harvest the cells (the same as above), discard the supernatant after centrifugation, resuspend with sodium chloride injection, adjust the final concentration to 5 x 10 6 cells / mL, and use it for subsequent experiments or preparation.

[0085] Example 2

[0086] The same as example 1, the main difference is that: 1.4 the culture medium in the culture of umbilical cord mesenchymal stem cells is different: the culture medium B is composed of 10 mL FBS + 90 mL α-MEM basic medium + 0.715 mL luteolin solution (437 μmol / L).

[0087] The luteolin solution is 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 same as example 1, the main difference is that: 1.4 the culture medium in the culture of umbilical cord mesenchymal stem cells is different: the culture medium C is composed of 10 mL FBS + 90 mL α-MEM basic medium + 1.116 mL of baicalin solution (280 μmol / L).

[0090] The baicalin solution is 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 same as example 1, the main difference is that: 1.4 the culture medium in the culture of umbilical cord mesenchymal stem cells is different: the culture medium D is composed of 10 mL FBS + 90 mL α-MEM basic 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 example 2.

[0094] The preparation of the baicalin solution is the same as example 3.

[0095] Example 5

[0096] The same as example 1, the main difference is that: 1.4 the culture medium in the culture of umbilical cord mesenchymal stem cells is different: the culture medium F is composed of 10 mL FBS + 90 mL α-MEM basic medium + 0.238 mL luteolin solution (437 μmol / L) + 0.372 mL baicalin solution (280 μmol / L) + 0.359 mL isobarreiroside solution (290 μmol / L).

[0097] The preparation of the luteolin solution is the same as example 2.

[0098] The preparation of the baicalin solution is the same as example 3.

[0099] The emodin solution was prepared as follows: 15 mg of emodin was dissolved in 0.2 mL of DMSO to obtain a 290 μmol / L emodin solution.

[0100] Comparative Example 1

[0101] The same as Example 1, the main difference is that: 1.4 The culture medium in the culture of umbilical cord mesenchymal stem cells is different: the culture medium E is composed of 10 mL of FBS + 90 mL of α-MEM basic medium + 1.078 mL of emodin solution (290 μmol / L).

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

[0103] Test Example

[0104] 1. CCK-8 detection of the effect of different culture medium additives on cell viability

[0105] Experimental process: cell recovery and preparation: the α-MEM basic medium was preheated to 37 ℃ for standby, and was loaded into a centrifuge tube for standby; the P4 generation of umbilical cord mesenchymal stem cells was recovered, and the cell suspension was transferred to a centrifuge tube containing 9 mL of preheated α-MEM basic medium, and was mixed thoroughly.

[0106] Cell suspension preparation: the above mixed cell suspension was equally divided into 6 centrifuge tubes, and was centrifuged at 1000 rpm for 5 min, and 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, and after being mixed thoroughly, 20 μL of the cell suspension was added to 20 μL of 0.2% trypan blue staining solution, and the viable cell count was performed.

[0107] 96-well plate inoculation: according to the cell count results, the concentration was adjusted to 40000 cells / mL with the corresponding culture medium, 100 μL of DPBS was added to each well of the 96-well plate, 100 μL of the cell suspension was taken and placed in the 96-well plate, and was placed in a 37 ℃, 5% CO2 incubator for 24 h, then 10 μL of CCK-8 reagent was precisely added to each well, and the incubation was continued for 2 h, the 96-well plate was taken out, and the absorbance was measured at 450 nm using an enzyme marker, and the corresponding cell number, viability and cell proliferation rate were calculated according to the absorbance, and the results are shown in Table 1. Figure 1 and Table 1.

[0108] The absorbance value was measured at 450 nm using an enzyme marker, and the difference in proliferation rate was calculated after subtracting the absorbance value of the blank control group from the absorbance value of each sample, and the difference in proliferation rate = the absorbance value measured at 450 nm of each example or comparative example / the absorbance value measured at 450 nm of Example 1 x 100%.

[0109] Table 1 cell proliferation difference rate

[0110]

[0111] From the above table, it can be seen that the cell proliferation difference rate of the stem cells prepared by the application in different culture media is relatively large. The addition of luteolin or baicalin alone can significantly improve the cell proliferation activity. Luteolin and baicalin are core synergistic components. When they are used together, the PI3K / Akt activation of luteolin and the VEGFR3-NFAT signal of baicalin form cross regulation. Through the activation of downstream PI3K / Akt, MAPK (ERK1 / 2) and other signal pathways, the endothelial cell proliferation (accelerating cell cycle progression) and migration (enhancing cell skeleton remodeling ability) are promoted. The double pathway synergistically promotes the expression of cell cycle proteins, and the double signal pathway synergistically activates cell growth. At the same time, the paracrine effect of PGE2 and VEGF-C further optimizes the microenvironment, resulting in the highest proliferation difference rate. The addition of isorhapontigenin alone has a weak promoting effect on proliferation. Isorhapontigenin is an auxiliary synergistic component, which further optimizes the proliferation effect by improving the microenvironment. When isorhapontigenin is used together with luteolin and baicalin, a multi-target network of anti-inflammatory (NF-κB inhibition), proliferation promotion (PI3K / Akt) and angiogenesis (VEGFR3 / Ang-1) is formed, which further reduces the cell apoptosis rate, and the proliferation difference rate is increased to 151.6%. This indicates that isorhapontigenin further optimizes the proliferation microenvironment of stem cells, and forms a triple synergistic effect with luteolin and baicalin, resulting in the largest proliferation difference rate.

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

[0113] Cell preparation: the α-MEM basic culture medium is preheated to 37 ℃ for standby, and is loaded into a centrifuge tube for standby; the P4 generation of umbilical cord mesenchymal stem cells is recovered, and the cell cryopreservation liquid is transferred to the centrifuge tube containing the preheated culture medium, and is fully mixed.

[0114] Cell inoculation and culture: the above mixed cell suspension is equally divided into 6 centrifuge tubes, and is centrifuged at a centrifugal speed of 1000 rpm for 5 min, and the supernatant is discarded. The cells are resuspended with 1 mL of the culture medium prepared in Examples 1-5 and Comparative Example 1, fully mixed, 20 μL of the cell suspension is added to 20 μL of 0.2% trypan blue staining solution, fully mixed, and the viable cell count is performed. According to the cell count result, the cells in different groups are inoculated into 6 culture dishes at a density of 7000 cells / cm 2Inoculate into T225 culture flask, add the culture medium prepared in Example 1-5 and Comparative Example 1, and place in a 37°C, 5% CO2 incubator for culture for 72 h. When the cell confluence reaches 90%, collect the cell supernatant to detect the secretion levels of PGE2 and VEGF-C.

[0115] 2.1, PGE2 detection: Prepare the required amount of reagent in advance, set up non-specific binding wells, blank control wells, standard and test samples. Add 200 μL of RD5-56 diluent to the non-specific binding wells (NSB); add 150 μL of diluent RD5-56 to the blank control wells (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 evenly with the gun for five times, seal the plate with sealing film, and incubate at room temperature with shaking at 550 rpm for 2 h. Add 50 μL of PGE2 Conjugate directly to each well, mix evenly with the gun for five times, seal the plate with sealing film, and incubate at room temperature with shaking at 500 rpm for 2.5 h. Discard the liquid in the wells, add 400 μL of Wash Buffer (1x) to each well, stand for 90 s, and repeat the plate washing four times. Pat dry the residual liquid. Add 200 μL of TMB substrate developing solution to each well, seal the plate with sealing film, and incubate at room temperature with shaking at 500 rpm for 30 min. Add 100 μL of 2 mol / L sulfuric acid to each well to stop the reaction, and read the absorbance value at 450 / 540 nm within 30 min on the microplate reader. The results are shown in Figure 2 .

[0116] 2.2, VEGF-C detection: Prepare the required amount of reagent as needed in advance, set up standard, positive control, blank control and test sample, and transfer 50 μL of diluted standard, positive control, blank control and test sample to the corresponding wells, respectively. Then add 100 μL / well of Assay Diluent RD1W diluent to each well, seal the plate with sealing film, and incubate at room temperature with shaking at 500 rpm for 2 h. After incubation, discard the liquid in the wells, add 400 μL of wash solution (1x) to each well, and repeat the plate washing four times. Pat dry the residual liquid. Add 200 μL of Human VEGF-C Conjugate HRP to each well, and incubate at room temperature with shaking at 500 rpm for 2 h. After washing, add 200 μL of TMB substrate developing solution to each well, seal the plate with sealing film, and incubate at room temperature in the dark for 30 min. Add 50 μL of 2 mol / L sulfuric acid to each well to stop the reaction, and read the absorbance value at 450 / 540 nm within 30 min on the microplate reader. The results are shown in Figure 3 .

[0117] 3. Animal experiment

[0118] Modeling of diabetic full-thickness skin defect model mice: 70 BKS-db male mice aged 8 weeks were purchased; all mice were adaptively fed for 1 week, and mice with fasting blood glucose ≥11.1 mmol / L were selected for the group. Hair on the back of the mice was removed using depilatory cream, and the back skin was disinfected using 75% alcohol and iodophor. A 10 mm diameter circular full-thickness skin defect wound was made on the back of the mice, and the wound was cared for on the same day of modeling. The successfully modeled mice were randomly divided into 7 groups, and the mice were administered according to Table 1:

[0119] Table 1: Mouse administration grouping

[0120]

[0121] Cell culture: α-MEM basal medium was preheated to 37 ℃ for standby, and was loaded into a centrifuge tube for standby; the P4 generation of umbilical cord mesenchymal stem cells was recovered, and the cell cryopreservation solution was transferred to the centrifuge tube containing the preheated culture medium, and was thoroughly mixed. The above-mentioned mixed cell suspension was equally divided into 6 centrifuge tubes, and was centrifuged at a centrifugal speed of 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with 1 mL of the medium prepared in Examples 1-5 and Comparative Example 1, and were thoroughly mixed. After 100 μL of the cell suspension was taken, 10-fold dilution was performed, and 20 μL of 0.2% trypan blue staining solution was thoroughly mixed, and viable cell counting was performed. According to the cell counting results, the cells in different groups were inoculated into four-layer cell factories at a concentration of 7000 cells / cm 2 The medium prepared in Examples 1-5 and Comparative Example 1 was added, and the cell factories were placed in a 37 ℃, 5% CO2 incubator for culture for 72 h. When the cell confluence reached 90%, the cells were harvested by digestion, the supernatant was discarded, the cells were resuspended with sodium chloride injection, and the concentration was adjusted to 5×10 6 cells / mL.

[0122] Mouse administration scheme: each mouse in the treatment group received a cell suspension injection;

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

[0124] Injection volume: 50 μL per point;

[0125] Total injection amount: 200 μL per mouse.

[0126] On days 0, 3, 7, 12, and 15, a standard scale was placed at each observation time, the wound healing was calculated, and wound photos were taken to record the healing, and the results are shown in Figure 4 and Figure 5 .

[0127] 4. Experimental result analysis:

[0128] Figure 1 Compared with group A, the number and activity of cells increased (P<0.05 at most time points) whether luteolin and baicalin were added alone or simultaneously, and the activity of umbilical cord mesenchymal stem cells was significantly improved. Among them, the activity of cells in group D with simultaneous addition of luteolin and baicalin increased most obviously (P<0.01), indicating that the two had a synergistic effect.

[0129] Figure 2 Among them, the addition of luteolin significantly promoted the secretion of PGE2, which was 1.84 times that of group A, and baicalin also had a promoting effect, which was 1.14 times that of group A. When luteolin and baicalin were added simultaneously, the secretion of PGE2 was the highest, which was 2.13 times that of group A. Literature shows that PGE2 acts as a self / paracrine signal factor for promoting proliferation and survival, and umbilical cord mesenchymal stem cells can produce and respond to it. PGE2 exerts its effect by binding to its G protein-coupled receptor on the cell surface. The applicant speculates that the addition of baicalin and luteolin may act on a common upstream regulatory point that can activate the proliferation pathway and induce the synthesis of PGE2.

[0130] Figure 3 Among them, the addition of baicalin significantly improved the secretion of VEGF-C, which was 1.57 times that of group A, and luteolin had a weak effect, which was 1.19 times that of group A. When luteolin and baicalin were added simultaneously, the effect was the best, which was 1.88 times that of group A. VEGF-C is a highly specific vascular endothelial cell growth factor that promotes increased vascular permeability, vascular endothelial cell migration, proliferation, and angiogenesis, and umbilical cord mesenchymal stem cells can produce and respond to it. After VEGF-C binds to VEGFR-2 / VEGFR-3 receptors on the surface of vascular endothelial cells, it activates downstream PI3K / Akt, MAPK (ERK1 / 2) signaling pathways, promotes endothelial cell proliferation (accelerates cell cycle progression), migration (enhances cytoskeleton remodeling ability), and induces endothelial cells to form a luminal structure (a key step in angiogenesis). In addition, VEGF-C can synergize with other pro-angiogenic factors (such as VEGF-A, 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 showed a promoting effect on wound healing relative to the blank control group at the D12 and D15 time points; relative to the culture group without additives (group A), the addition of baicalin or luteolin alone showed a higher healing rate at the D12 and D15 time points; and the combined addition of baicalin and luteolin (group D) showed the highest wound healing rate at each detection time point, with a healing rate of 82% at D12 (the healing rate range of no addition or addition of only one additive was 32%-76%) and a healing rate of up to 96% at D15 (the healing rate range of no addition or addition of only one additive was 36%-82%).

[0132] Figure 5 In the experiment, the blank group showed delayed wound healing, accompanied by obvious inflammation and edge contraction; group A only partially improved, while the re-epithelialization of the wound was faster when luteolin and baicalin were added together (group D), and the results were consistent with Figure 4 The data are consistent, and the combined addition of the two additives enhances the viability of umbilical cord mesenchymal stem cells in mice, promoting faster wound healing.

[0133] Figure 6 In the experiment, the tissue at the wound site of the animals in groups B, C, and D was taken at D15, and HE staining was performed, and the granulation tissue and inflammatory cell infiltration were observed under a microscope. The granulation tissue of the group in which luteolin and baicalin were added together (group D) was higher than that of the group in which luteolin was added alone (group B, P<0.05) or the group in which baicalin was added alone (group C, P>0.05); and the inflammatory level at the wound site was lower than that of the groups in which luteolin or baicalin was added alone. The stem cells cultured with the addition of luteolin and baicalin together may inhibit local inflammation, regulate immunity to create an "anti-inflammatory microenvironment", promote angiogenesis to improve the "blood supply microenvironment", and ultimately enhance the tissue repair capacity of umbilical cord mesenchymal stem cells, possibly through the high secretion of PGE2 and VEGF-C.

[0134] In summary, the addition of baicalin and luteolin can improve the cell viability and proliferation capacity 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, and further enhance the ability of umbilical cord mesenchymal stem cells to promote angiogenesis and inhibit inflammation, thereby significantly promoting 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, baicalin, and isodanin are added as culture medium additives to a serum-supplemented culture medium; wherein the concentration of luteolin is 1.0301 μmol / L, the concentration of baicalin is 1.0316 μmol / L, and the concentration of isodanin is 1.0311 μmol / L; the cultured mesenchymal stem cells promote angiogenesis and lymphangiogenesis by activating the VEGFR3-CaN-NFAT, EP3-PI3K / Akt, and Akt-eNOS signaling pathways; the stem cells are umbilical cord-derived mesenchymal stem cells.

2. The method for culturing stem cells to promote wound healing according to claim 1, wherein: The steps include: Step 1: Providing umbilical cord-derived mesenchymal stem cells; Step 2: adding luteolin, baicalin, and isodanin 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.

3. The method for culturing stem cells to promote wound healing according to claim 2, wherein: The steps include: Step 1: Providing umbilical cord-derived mesenchymal stem cells; Step 2: adding luteolin, baicalin, and isodanin as culture medium additives to the serum-supplemented culture medium, wherein the concentration of luteolin is 1.0301 μmol / L, the concentration of baicalin is 1.0316 μmol / L, and the concentration of isodanin is 1.0311 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.

4. The method for culturing stem cells to promote wound healing according to claim 2 or 3, 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.

5. The method for culturing stem cells for promoting wound healing according to any one of claims 1 to 3, 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%.

6. The method for culturing stem cells to promote wound healing according to claim 2 or 3, wherein: The mesenchymal stem cells in step 4 are stem cells at passage P4-P8.

7. The stem cell culture method for promoting wound healing according to claim 2 or 3, characterized in that: The factors secreted by the cultured mesenchymal stem cells include VEGF-C and PGE2.

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