A skin repair solution containing stem cell factors and preparation method thereof

Abstract

The invention provides a skin repair liquid containing stem cell factors and a preparation method thereof. The skin repair liquid comprises, by weight, 8 to 12 parts of stem cell factors, 30 to 40 parts of chitosan hydrogel, 12 to 16 parts of Centella asiatica extract, 4 to 8 parts of Coptis chinensis extract, 4 to 12 parts of polyethylene glycol, and the balance is deionized water. The skin repair liquid has high safety and biocompatibility, is non-irritating and has no side effects, is suitable for people of various skin types and age groups, has the advantages of comprehensive repair, strong pertinence, significant effect and high safety, can meet the needs of burn skin repair, and provides a new choice for burn patients.

Description

Technical Field

[0001] The invention belongs to the technical field of skin repair, and specifically relates to a skin repair liquid containing stem cell factors and a preparation method thereof. Background Art

[0002] Burns are a common traumatic disease that often causes serious damage to the skin and other tissues, and even threatens the patient's life. In the treatment of burns, how to effectively promote wound healing, reduce scar formation, and reduce the risk of infection has always been the focus of medical attention.

[0003] Traditional treatments for burns mainly include medication, surgery, and physical therapy. However, these methods have many limitations in terms of treatment effect, recovery speed, and patient comfort. For example, although medication can inhibit inflammation and reduce infection, the side effects and drug resistance of the drugs cannot be ignored. Although surgery can directly repair damaged tissues, it is traumatic, takes a long time to recover, and has the risk of infection. Although physical therapy can improve local blood circulation and promote tissue repair, the treatment effect is unstable and it is difficult to apply to all types of burns.

[0004] With the rapid development of stem cell technology, its application in the field of burn treatment has gradually received attention. Stem cells have the ability of self-renewal and multidirectional differentiation, can differentiate into various types of cells, and participate in tissue repair and regeneration. Stem cell-based treatment methods are considered to be a potential burn treatment method; however, most of the stem cell treatment products currently on the market have problems such as high cost, complex preparation, and poor stability, making them difficult to be widely used in clinical practice. The application of stem cell therapy in the field of burns is still in its early stages, and related research and technologies need to be further improved and optimized. Summary of the invention

[0005] The present invention provides a skin repair liquid containing stem cell factor and a preparation method thereof, with the purpose of providing a repair liquid with stem cell factor as a main component combined with other plant extracts having skin repair-promoting and anti-inflammatory effects, in order to achieve better treatment effects and patient experience in burn treatment.

[0006] The scheme of the present invention is as follows:

[0007] In a first aspect, the present invention provides a skin repair liquid containing stem cell factor, which comprises, by mass, 8 to 12 parts of stem cell factor, 30 to 40 parts of chitosan hydrogel, 12 to 16 parts of Centella asiatica extract, 4 to 8 parts of Coptis chinensis extract, 4 to 12 parts of polyethylene glycol, and the balance is deionized water.

[0008] Furthermore, the stem cell factor is taken from human umbilical cord stem cells; the total content of the effective ingredients in the Centella asiatica extract: asiatica glycoside and hydroxymadecassoside is not less than 50 μg / g; the mass proportion of berberine in the Coptis chinensis extract is not less than 20%.

[0009] The mechanism of action of the above raw material components is as follows:

[0010] Coptis chinensis extract, Centella asiatica extract and human stem cells each have unique mechanisms and effects in treating burns. When they are used together to treat burns, their mechanisms will synergize and enhance each other; Coptis chinensis extract has anti-inflammatory, antipyretic, analgesic, antibacterial and choleretic effects. Its anti-inflammatory effect is particularly important in treating burns, because skin tissue often undergoes inflammatory reactions after burns, leading to symptoms such as redness, swelling and pain. Alkaloids such as coptisine in Coptis chinensis extract can inhibit the release of inflammatory mediators and the expression of cytokines, thereby reducing symptoms such as tissue redness and swelling, and helping to relieve inflammatory reactions after burns; Centella asiatica extract has the effects of clearing away heat and dampness, detoxifying and reducing swelling, promoting blood circulation and removing blood stasis, anti-inflammatory and analgesic, and promoting wound healing. In the treatment of burns, the anti-inflammatory, analgesic and wound healing effects of Centella asiatica extract are very critical. It can inhibit inflammatory reactions and reduce the transmission of pain signals, thereby reducing the pain after burns.

[0011] At the same time, Centella asiatica extract can also promote wound healing, accelerate the repair and regeneration of damaged tissues, and help burn wounds recover quickly. When human stem cells are used to treat burns, they can differentiate into damaged skin cells and replace necrotic or damaged tissues, thereby accelerating the healing and repair of burn wounds. In addition, stem cells can also regulate the immune system, reduce inflammatory reactions, improve the immunity of burn patients, and help prevent complications such as infection.

[0012] When Coptis chinensis extract, Centella asiatica extract and human stem cells are used together to treat burns, their mechanisms will synergize and enhance each other. Coptis chinensis extract and Centella asiatica extract can inhibit inflammatory response, relieve pain, promote wound healing, etc., while stem cells can differentiate into damaged skin cells to accelerate wound healing and repair. This combination treatment may make the recovery of burn wounds faster and more effective, while reducing patients' pain and discomfort.

[0013] In a second aspect, the present invention provides a method for preparing a skin repair solution containing stem cell factors, which is used to prepare the skin repair solution described in the first aspect, and the preparation method comprises the following steps:

[0014] Step S1, putting stem cell factor, Centella asiatica extract, Coptis chinensis extract, polyethylene glycol and deionized water into a container and stirring for 5 to 10 minutes to obtain a mixture.

[0015] Step S2, adding chitosan hydrogel into the container, stirring evenly so that the mixture is immersed in the chitosan hydrogel, standing for 4 to 6 hours, stirring again for 15 to 30 minutes, and obtaining a skin repair solution containing stem cell factors.

[0016] Chitosan hydrogel has good water absorption and moisturizing properties. It can absorb and stabilize the effective ingredients in the repair solution, slowly release and act on damaged skin. Chitosan hydrogel is a new type of biomaterial, which is composed of chitosan and other biomaterials, and has good biocompatibility and bioactivity. Chitosan is a natural polymer material with biological activities such as antibacterial, anti-inflammatory and wound healing. Therefore, chitosan hydrogel can be used in drug delivery, tissue engineering and wound healing. On this basis, the design of the layered hydrogel makes it have different mechanical, electromagnetic and chemical properties from the top layer to the bottom layer. This design enables the hydrogel to provide customized repair solutions according to different skin layers and repair needs. The hydrogel on the top layer may focus more on moisturizing and anti-inflammatory effects, while the hydrogel on the bottom layer may focus more on promoting the regeneration and repair of skin cells. The effect of chitosan hydrogel on the skin.

[0017] Furthermore, the chitosan hydrogel is a layered structure, and its preparation method comprises the following steps:

[0018] Step S11. Weigh chitosan powder and 2 vol% acetic acid solution to prepare a 4 wt% chitosan / acetic acid solution and record it as the first mixed solution. Stir for 4 hours and let it stand for 24 hours to remove bubbles. The chitosan powder should be evenly dispersed in the acetic acid solution to avoid forming lumps. Chitosan dissolves in acetic acid to form a stable solution.

[0019] Step S12. Pour the first mixed solution into a template test tube, shake the template test tube to form a uniform thin film of chitosan / acetic acid solution on its inner surface, then pour out the remaining chitosan / acetic acid solution, and then soak the thin film template test tube with uniform chitosan / acetic acid solution in 3-5wt% NaOH solution for 2h, so that the thin film of chitosan / acetic acid solution is physically cross-linked under alkaline conditions.

[0020] Step S13. Inject chitosan / acetic acid solution into the film template test tube containing chitosan / acetic acid solution again, soak the template test tube in 3-5wt% NaOH solution for 4 hours, and then place the template test tube in deionized water for 2 hours to separate the template test tube from the cross-linked chitosan / acetic acid solution to obtain a chitosan hydrogel rod.

[0021] Step S14. Alternately soak the chitosan hydrogel stick in a 3-5wt% NaOH solution for 2 hours and in deionized water for 2 hours. Repeat the alternating process 4-6 times, and then rinse the chitosan hydrogel stick in deionized water until it becomes neutral.

[0022] Step S15. Place the chitosan hydrogel sticks prepared by the alternating immersion method in a drying oven at 55-60°C and turn them over every 30-60 minutes. After drying for 10 hours, take them out and weigh them every 10 minutes. Stop drying when there is no change in the measured mass.

[0023] Step S16. Place the dried chitosan hydrogel stick in deionized water at 20° C. for 30 h, and wipe off excess surface water with filter paper to obtain a chitosan hydrogel with a layered structure.

[0024] It should be noted that when soaking in NaOH solution, the temperature and time must be accurately controlled. The temperature should be controlled within 30°C to avoid excessive degradation of chitosan. The hydrogel sticks should be turned over regularly during the drying process to ensure uniform drying. In NaOH solution, the amino groups on the chitosan molecular chain undergo ion exchange reactions with the hydroxide ions of NaOH to form a stable gel network. Alternating soaking in NaOH solution and deionized water can further adjust the crosslinking degree and stability of the gel.

[0025] Furthermore, the preparation method of the Centella asiatica extract comprises: crushing the dried Centella asiatica, adding 15 to 25 times by weight of deionized water, adding ethanol in an amount equal to that of the deionized water at a temperature of 40 to 50° C., and stirring for 8 to 12 hours to obtain an extract of asiatica glycoside and madecassoside;

[0026] The extract is filtered, decolorized with activated carbon, and then concentrated and dried to obtain the Centella asiatica extract.

[0027] Centella asiatica is a traditional Chinese medicine with multiple functions such as clearing away heat and dampness, detoxifying and reducing swelling. Its extracts are mainly madecassoside and madecassoside, polyacetylene olefins, volatile oils and other chemical components. These ingredients in Centella asiatica extract have multiple skin care effects; they can inhibit the activity of tyrosinase, reduce the production and deposition of melanin, and thus achieve a certain whitening effect. These active ingredients in Centella asiatica extract also have strong antioxidant effects, which can inhibit the activity of free radicals, protect skin cells from oxidative damage, and keep the skin smooth and tender.

[0028] In addition, the ingredients in Centella asiatica extract also have the function of repairing skin damage and inhibiting scar proliferation. They can act on fibroblasts, increase the synthesis of type I and type II collagen inside and outside cells, upregulate the genes related to skin damage repair, and increase the synthesis of extracellular matrix to accelerate the process of skin damage repair. At the same time, they can also affect the ultrastructure of fibroblasts, making the proliferation of fibroblasts inactive, thereby inhibiting scar proliferation.

[0029] Centella asiatica has a certain degree of water solubility, and these ingredients can dissolve or disperse in the aqueous environment of the hydrogel, thus forming good compatibility with the hydrogel. In addition, the ingredients of Centella asiatica and hydroxy-madecassoside also interact with the polymer chains in the hydrogel. The Centella asiatica and stem cell factor are co-loaded in the chitosan hydrogel, which can give full play to the medicinal value of Centella asiatica and the regenerative function of stem cell factor, and with the help of the sustained release and targeted delivery of chitosan hydrogel, the layered treatment effect can be achieved while reducing side effects.

[0030] Furthermore, the preparation method of the stem cell factor comprises:

[0031] Step S21, aseptically collecting the umbilical cord of the newborn, cutting the tissue into pieces, and ensuring that each piece of tissue is of uniform size.

[0032] Step S22, culturing by direct tissue culture method, placing the tissue in a fresh serum-free culture medium containing an enzymatic hydrolysis solution, wherein the enzymatic hydrolysis solution is pancreatin or collagenase.

[0033] Step S23, performing enzymatic hydrolysis in a constant temperature incubator, and diluting the enzymatic hydrolyzate to 3 to 5 times with a culture medium after the enzymatic hydrolysis.

[0034] Step S24, removing residual tissue fragments by filtering and centrifuging to obtain a cell suspension.

[0035] Step S25, inoculating the cell suspension into a culture vessel for culture, adding fresh serum-free culture medium to continue passage to obtain trophoblast fluid.

[0036] Step S26, add the trophoblast fluid to DMEM / F12 culture medium containing 15% FBS by volume, place in a 37°C, 5% CO 2 Culture in a saturated humidity incubator, add fresh culture medium every 3 days, at a rate of 1×10 4 cell / cm 2 The culture medium was replaced with DMEM / F12 medium containing 10% FBS by volume, and the cell generation was recorded as P1. After the cells reached 70-90% confluence, they were digested and cultured at 1×10 4 cell / cm 2 The cell generation number was recorded as P2 after subculture.

[0037] Step S27, collect P2 cells, wash the cells with D-Hank's balanced salt solution, grind them by centrifugation and dry them to obtain the desired stem cell factors.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention realizes comprehensive repair of burned skin by combining multiple ingredients such as stem cell factor, chitosan hydrogel, Centella asiatica extract, Coptis chinensis extract and polyethylene glycol. The repair liquid can quickly promote the healing of burned skin, reduce scar formation, and restore the color, texture and elasticity of the skin. The ingredients in the repair liquid are all from natural substances or human cells, have high safety and biocompatibility, are non-irritating and have no side effects, are suitable for people of various skin types and age groups, have the advantages of comprehensive repair, strong targeting, significant effect and high safety, can meet the needs of burned skin repair, and provide a new choice for burn patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a SEM image of a skin repair solution containing stem cell factors according to the present invention.

[0041] Figure 2 The present invention is a flow chart of a method for preparing a skin repair solution containing stem cell factors. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] A skin repair solution containing stem cell factors, composed of the following ingredients:

[0045] The skin repair liquid includes, by weight: 8 to 12 parts of stem cell factor, 30 to 40 parts of chitosan hydrogel, 12 to 16 parts of Centella asiatica extract, 4 to 8 parts of Coptis chinensis extract, 4 to 12 parts of polyethylene glycol, and the balance is deionized water.

[0046] The stem cell factor is taken from human umbilical cord stem cells; the total content of the effective ingredients in the Centella asiatica extract: asiatica glycoside and hydroxy-madecassoside is not less than 50 μg / g; the mass proportion of berberine in the Coptis chinensis extract is not less than 20%.

[0047] Figure 1 This is the SEM image of the skin repair fluid containing stem cell factors of this embodiment. From the SEM image, it can be seen that the larger circular area in the middle is the stem cells themselves captured by SEM in the skin repair fluid, which remain round in shape and have plump cytoplasm. During the treatment process, when the skin repair fluid is applied to the damaged skin surface, the stem cell factors and the existing exosome components will quickly penetrate into the deep layers of the skin and interact with the damaged cells. The skin repair fluid can significantly improve the condition of damaged skin by promoting cell regeneration and repair, reducing scar formation, increasing skin elasticity and firmness, and alleviating signs of skin aging.

[0048] Example 2

[0049] The method for preparing a skin repair solution containing stem cell factors in Example 1 is as follows Figure 2 As shown, the specific operations are as follows:

[0050] Step S1, putting stem cell factor, Centella asiatica extract, Coptis chinensis extract, polyethylene glycol and deionized water into a container and stirring for 5 to 10 minutes to obtain a mixture.

[0051] Step S2, adding chitosan hydrogel into the container, stirring evenly so that the mixture is immersed in the chitosan hydrogel, standing for 4 to 6 hours, stirring again for 15 to 30 minutes, and obtaining a skin repair solution containing stem cell factors.

[0052] It should be noted that stem cell factors are obtained through the cultivation of human umbilical cord tissue. The operating procedures for collecting human umbilical cords are as follows: Before collecting the umbilical cord, the collector must strictly check the name, age, and ID card information of the mother, and whether the mother is infected with hepatitis B, hepatitis C, AIDS, or syphilis. Only those who meet the preservation standards can have the umbilical cord collected.

[0053] The umbilical cord collection bottle must be a sterile container approved for human tissue collection to reduce the risk of cell loss and microbial contamination. Check whether the collection bottle is complete, airtight, and sterile, and whether the outer packaging is damaged. If damaged, it cannot be used. All reagents and materials used to collect the umbilical cord must be sterile.

[0054] Within 10 seconds after the newborn is delivered, clamp the umbilical cord 5-8 cm away from the newborn's navel with two hemostatic forceps, cut the umbilical cord between the two forceps and ligate it. Within 1 minute after the placenta is delivered, ligate the umbilical cord close to the placenta and then cut it. Ensure that the total length of the collected umbilical cord is not less than 15 cm, and the umbilical cord remains intact without needle holes or damage. Try to avoid the umbilical cord from contact with other objects.

[0055] Use an ordinary sterile tube to collect 5 ml of umbilical cord blood for pathogen detection. Wipe the ligated umbilical cord twice with sterile medical gauze soaked in 0.9% saline to remove blood, meconium and other dirt. Put the umbilical cord into a disposable sterile collection bottle and tighten the bottle cap.

[0056] The umbilical cord collection bottle, anticoagulant tube, and collection registration form should be uniformly labeled and placed in an outer packaging bag. After the umbilical cord is collected, it should be temporarily stored in a 4°C incubator and quickly transported to the laboratory.

[0057] The stem cell factor preparation process is as follows: The experimenter should carefully check whether the outer packaging of the sample is qualified and whether there is any damage, whether the umbilical cord bottle is airtight, whether there is any liquid spillage, whether the blood sample in the blood collection tube meets the test standards, and check whether the sample label content is consistent with the sample. Carefully check the basic information of the umbilical cord and keep good experimental records.

[0058] The experimental equipment and reagents required for umbilical cord separation include: autoclaved surgical box (including single scissors, tweezers, tissue scissors, gauze), autoclaved glass pipette, sterilized plastic tube, vacuum pump, φ10cm sterile culture dish, disposable sterile pipette, 50ml centrifuge tube, PBS, culture medium, complete culture medium.

[0059] The preparation method of stem cell factor comprises:

[0060] Step S21, aseptically collect the umbilical cord of the newborn, cut the tissue into pieces, ensure that each piece of tissue is of uniform size, cut off the ligatures at both ends of the umbilical cord, select the umbilical cord with good brightness, no damage, and no edema, cut it into several small pieces of about 1-2 cm, and carefully and repeatedly clean the impurities and pollutants on the outer surface of the umbilical cord with PBS to wash away the blood remaining in the blood vessels.

[0061] Step S22, culturing by direct tissue culture method, placing the tissue in a fresh serum-free culture medium containing an enzymatic hydrolysis solution, wherein the enzymatic hydrolysis solution is pancreatin or collagenase.

[0062] In a clean bench, add 6-8 ml of culture medium to a φ10 cm culture dish, place the dish on ice, cut the umbilical cord into 1 mm3 pieces as quickly as possible (within 30 min) using special scissors for umbilical cord tissue, add fresh serum-free culture medium containing enzymatic solution (trypsin or collagenase), and place at 37°C and 5% CO. 2 Culture in an incubator (try not to shake the culture dish within 24 hours).

[0063] Step S23, perform enzymatic hydrolysis in a constant temperature incubator, and dilute the enzymatic hydrolyzate to 3-5 times with culture medium after enzymatic hydrolysis: carefully observe the tissue block under a microscope to see if there are any signs of contamination, and replenish the fluid if there are no abnormalities. Gently add 18-40 ml of fresh complete culture medium to each φ10 cm culture dish and place it in a 37°C, 5% CO2 incubator for culture.

[0064] Step S24, removing residual tissue fragments by filtering and centrifuging to obtain a cell suspension.

[0065] Step S25, inoculating the cell suspension into a culture vessel for culture, adding fresh serum-free culture medium to continue passage to obtain trophoblast fluid.

[0066] Step S26, add the trophoblast fluid to DMEM / F12 culture medium containing 15% FBS by volume, place in a 37°C, 5% CO 2 Culture in a saturated humidity incubator, add fresh culture medium every 3 days, and adjust the concentration of 1×10 4 cell / cm 2 The culture medium was replaced with DMEM / F12 medium containing 10% FBS by volume, and the cell generation was recorded as P1. After the cells reached 70-90% confluence, they were digested and cultured at 1×10 4 cell / cm 2 The cell generation number was recorded as P2 after subculture.

[0067] Step S27, collect P2 cells, wash the cells with D-Hank's balanced salt solution, grind them by centrifugation and dry them to obtain the desired stem cell factors.

[0068] The chitosan hydrogel is a layered structure, and its preparation method comprises the following steps:

[0069] Step S11. Weigh chitosan powder and 2 vol% acetic acid solution to prepare a 4 wt% chitosan / acetic acid solution and record it as the first mixed solution. Stir for 4 hours and let it stand for 24 hours to remove bubbles. The chitosan powder should be evenly dispersed in the acetic acid solution to avoid forming lumps. Chitosan dissolves in acetic acid to form a stable solution.

[0070] Step S12. Pour the first mixed solution into a template test tube, shake the template test tube to form a uniform thin film of chitosan / acetic acid solution on its inner surface, then pour out the remaining chitosan / acetic acid solution, and then soak the thin film template test tube with uniform chitosan / acetic acid solution in 3-5wt% NaOH solution for 2h, so that the thin film of chitosan / acetic acid solution is physically cross-linked under alkaline conditions.

[0071] Step S13. Inject chitosan / acetic acid solution into the film template test tube containing chitosan / acetic acid solution again, soak the template test tube in 3-5wt% NaOH solution for 4 hours, and then place the template test tube in deionized water for 2 hours to separate the template test tube from the cross-linked chitosan / acetic acid solution to obtain a chitosan hydrogel rod.

[0072] Step S14. Alternately soak the chitosan hydrogel stick in a 3-5wt% NaOH solution for 2 hours and in deionized water for 2 hours. Repeat the alternating process 4-6 times, and then rinse the chitosan hydrogel stick in deionized water until it becomes neutral.

[0073] Step S15. Place the chitosan hydrogel sticks prepared by the alternating immersion method in a drying oven at 55-60°C and turn them over every 30-60 minutes. After drying for 10 hours, take them out and weigh them every 10 minutes. Stop drying when there is no change in the measured mass.

[0074] Step S16. Place the dried chitosan hydrogel stick in deionized water at 20° C. for 30 h, and wipe off excess surface water with filter paper to obtain a chitosan hydrogel with a layered structure.

[0075] The preparation method of Centella asiatica extract includes: crushing the dried Centella asiatica, adding 15 to 25 times the mass of deionized water, adding ethanol in an amount equal to the mass of the deionized water at a temperature of 40 to 50°C, stirring for 8 to 12 hours to obtain an extract of Centella asiatica glycoside and hydroxy-madecassoside; filtering the extract, decolorizing it with activated carbon, concentrating and drying it to obtain the Centella asiatica extract.

[0076] Example 3

[0077] Clinical effect verification:

[0078] From March to December 2023, the above-mentioned stem cell factor skin repair fluid was used to treat 300 patients with local burns. The burn area was no more than 5 cm2. The causes were mostly flames, high-temperature liquids, steam, electricity, chemicals, etc. in contact with the skin or exposure to high-temperature environments, resulting in damage to the skin and other tissues that may be involved.

[0079] Test cases: 300 cases, aged 12 to 60 years old, with an average age of 34 years old.

[0080] Treatment method: Take the stem cell factor skin repair liquid of Example 1, apply it evenly to the part to be repaired, and massage for 3 to 5 minutes, preferably 2 to 4 times a day. After opening, it should be refrigerated and used up within ten days as much as possible.

[0081] Two thirds of the patients were successfully treated with the skin repair solution containing stem cell factors in Example 1:

[0082] From the 1st to the 15th day, during the rapid penetration and deep nourishment period, multiple active factors are rapidly absorbed, promoting cell regeneration, the wounds at the patient's burn site gradually shrink, necrotic tissue is effectively removed, and new granulation tissue begins to grow. The wound surface gradually becomes dry, with no obvious signs of exudation and infection. As the treatment progresses, the pain at the patient's burn site gradually subsides.

[0083] On the 15th to 30th day, collagen is replenished and the skin undergoes regeneration. The skin at the burned area gradually heals, the color of the new skin approaches normal, and the redness and swelling of the skin around the burn gradually decreases.

[0084] On the 30th to 45th day, the skin becomes further firmed and smoothed, with a soft texture, and the redness and swelling of the skin around the burn gradually decreases and disappears.

[0085] Comparison group: 200 cases, aged 15 to 56 years old, with an average age of 31 years old.

[0086] Use the stem cell factor skin repair solution of Example 1 which is different from that used in the test example, but does not contain Centella asiatica extract and Coptis chinensis extract. Apply it evenly to the area to be repaired and massage for 3 to 5 minutes, preferably 2 to 4 times a day. After opening, refrigerate and use up within ten days as much as possible.

[0087] One quarter of the patients were successfully treated with the control group of skin repair solutions containing stem cell factors:

[0088] Day 1-15: In the early stage of treatment, the wound began to shrink, but the speed was slower than that in the test case. The removal of necrotic tissue and the growth of new granulation tissue were not as significant as in the test case. Although the wound surface began to dry, there was still a small amount of exudation and slight signs of infection. The patient's pain was relieved, but the degree of relief was not as significant as in the test case.

[0089] Day 15-30: Entering the collagen replenishment and skin regeneration phase, the patient's burnt skin began to heal, but the color and texture of the new skin were somewhat different from those of the test case. The new skin was darker in color and harder in texture, not as close to normal skin as the new skin in the test case. The redness and swelling of the skin around the burn was reduced, but the reduction rate was slow.

[0090] Day 30-45: The patient's skin firmness and smoothness improved, but the effect was limited compared to the test case. Although the redness and swelling of the skin around the burn gradually decreased, there was still a certain degree of redness and swelling.

[0091] Conclusion: Through comparative experiments, we found that the skin repair solution containing stem cell factor, Centella asiatica extract and Coptis chinensis extract in the test case is significantly better than the repair solution without these two plant extracts in terms of therapeutic effect. The addition of Centella asiatica extract and Coptis chinensis extract not only accelerates the wound healing process, but also improves the quality and color of the new skin and reduces the pain of the patient. This result further proves the scientificity and effectiveness of the repair solution formula in the test case.

[0092] The present invention is described by specific embodiments, and it should be understood by those skilled in the art that various changes and equivalent substitutions may be made to the present invention without departing from the scope of the present invention. In addition, various modifications may be made to the present invention for specific situations or materials without departing from the scope of the present invention. Therefore, the present invention is not limited to the disclosed specific embodiments, but should include all implementation methods falling within the scope of the claims of the present invention.

Claims

1. A skin repair solution containing stem cell factors, characterized in that: The skin repair solution comprises, by weight: 8 to 12 parts of stem cell factor, 30 to 40 parts of chitosan hydrogel, 12 to 16 parts of Centella asiatica extract, 4 to 8 parts of Coptis chinensis extract, 4 to 12 parts of polyethylene glycol, and the balance is deionized water; The chitosan hydrogel is a layered structure, and its preparation method comprises the following steps: Step S11. Weigh chitosan powder and 2 vol% acetic acid solution to prepare a 4 wt% chitosan / acetic acid solution and record it as the first mixed solution. Stir for 4 hours and then let it stand for 24 hours to remove bubbles. The chitosan powder should be evenly dispersed in the acetic acid solution to avoid the formation of lumps. The chitosan is dissolved in the acetic acid to form a stable solution. Step S12. pouring the first mixed solution into a template test tube, shaking the template test tube to form a uniform chitosan / acetic acid solution film on its inner surface, then pouring out the remaining chitosan / acetic acid solution, and then soaking the template test tube with the uniform chitosan / acetic acid solution film in a 3-5wt% NaOH solution for 2h, so that the chitosan / acetic acid solution film is physically cross-linked under alkaline conditions; Step S13. injecting chitosan / acetic acid solution into the film template test tube containing chitosan / acetic acid solution again, soaking the template test tube in 3-5wt% NaOH solution for 4 hours, and then placing the template test tube in deionized water for 2 hours to separate the template test tube from the cross-linked chitosan / acetic acid solution to obtain a chitosan hydrogel rod; Step S14. Alternately soak the chitosan hydrogel stick in a 3-5wt% NaOH solution for 2 hours and in deionized water for 2 hours, repeat the alternating process 4-6 times, and then rinse the chitosan hydrogel stick in deionized water until it becomes neutral; Step S15. Place the chitosan hydrogel sticks prepared by the alternating immersion method in a drying oven at 55-60°C and turn them over every 30-60 minutes. After drying for 10 hours, take them out and weigh them every 10 minutes. Stop drying when there is no change in the measured mass. Step S16. Place the dried chitosan hydrogel stick in deionized water at 20° C. for 30 h, and wipe off excess surface water with filter paper to obtain a chitosan hydrogel with a layered structure.

2. The skin repair solution containing stem cell factors according to claim 1, characterized in that: The stem cell factor is taken from human umbilical cord stem cells.

3. The skin repair solution containing stem cell factors according to claim 2, characterized in that: The total content of the effective components in the Centella asiatica extract: asiatica glycoside and madecassoside is not less than 50 μg / g.

4. The skin repair solution containing stem cell factors according to claim 3, characterized in that: The mass percentage of berberine in the Coptis chinensis extract is not less than 20%.

5. A method for preparing a skin repair solution containing stem cell factors, for preparing the skin repair solution according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Step S1, placing stem cell factor, Centella asiatica extract, Coptis chinensis extract, polyethylene glycol and deionized water in a container and stirring for 5 to 10 minutes to obtain a mixture; Step S2, adding chitosan hydrogel into the container, stirring evenly so that the mixture is immersed in the chitosan hydrogel, standing for 4 to 6 hours, stirring again for 15 to 30 minutes, and obtaining a skin repair solution containing stem cell factors.

6. The method for preparing a skin repair solution containing stem cell factors according to claim 5, characterized in that: The chitosan hydrogel is a layered structure, and its preparation method comprises the following steps: Step S11. Weigh chitosan powder and 2 vol% acetic acid solution to prepare a 4 wt% chitosan / acetic acid solution and record it as the first mixed solution, stir for 4 hours and then stand for 24 hours to remove bubbles; Step S12. pouring the first mixed solution into a template test tube, shaking the template test tube to form a uniform chitosan / acetic acid solution film on its inner surface, then pouring out the remaining chitosan / acetic acid solution, and then soaking the template test tube with the uniform chitosan / acetic acid solution film in a 3-5wt% NaOH solution for 2h, so that the chitosan / acetic acid solution film is physically cross-linked under alkaline conditions; Step S13. injecting chitosan / acetic acid solution into the film template test tube containing chitosan / acetic acid solution again, soaking the template test tube in 3-5wt% NaOH solution for 4 hours, and then placing the template test tube in deionized water for 2 hours to separate the template test tube from the cross-linked chitosan / acetic acid solution to obtain a chitosan hydrogel rod; Step S14. Alternately soak the chitosan hydrogel stick in a 3-5wt% NaOH solution for 2 hours and in deionized water for 2 hours, repeat the alternating process 4-6 times, and then rinse the chitosan hydrogel stick in deionized water until it becomes neutral; Step S15. Place the chitosan hydrogel sticks prepared by the alternating immersion method in a drying oven at 55-60°C and turn them over every 30-60 minutes. After drying for 10 hours, take them out and weigh them every 10 minutes. Stop drying when there is no change in the measured mass. Step S16. Place the dried chitosan hydrogel stick in deionized water at 20° C. for 30 h, and wipe off excess surface water with filter paper to obtain a chitosan hydrogel with a layered structure.

7. The method for preparing a skin repair solution containing stem cell factors according to claim 6, characterized in that: The preparation method of the Centella asiatica extract comprises: crushing the dried Centella asiatica, adding 15 to 25 times by weight of deionized water, adding ethanol in an amount equal to the amount of deionized water at a temperature of 40 to 50° C., and stirring for 8 to 12 hours to obtain an extract of asiatica glycoside and madecassoside; The extract is filtered, decolorized with activated carbon, and then concentrated and dried to obtain the Centella asiatica extract.

8. The method for preparing the skin repair solution containing stem cell factors according to claim 6, characterized in that: The preparation method of the stem cell factor comprises: Step S21, aseptically collecting the umbilical cord of the newborn, cutting the tissue into pieces, and ensuring that each piece of tissue is of uniform size; Step S22, culturing by direct tissue culture method, placing the tissue in a fresh serum-free culture medium containing an enzymatic hydrolyzate, wherein the enzymatic hydrolyzate is pancreatin or collagenase; Step S23, performing enzymatic hydrolysis in a constant temperature incubator, and diluting the enzymatic hydrolyzate to 3 to 5 times with a culture medium after the enzymatic hydrolysis; Step S24, removing residual tissue fragments by filtering and centrifuging to obtain a cell suspension; Step S25, inoculating the cell suspension in a culture vessel for culture, adding fresh serum-free culture medium to continue passage to obtain trophoblast cell fluid; Step S26, add the trophoblast fluid to DMEM / F12 culture medium containing 15% FBS by volume, place in a 37°C, 5% CO 2 Culture in a saturated humidity incubator, add fresh culture medium every 3 days, at a rate of 1×10 4 cell / cm 2 The culture medium was replaced with DMEM / F12 medium containing 10% FBS by volume, and the cell generation was recorded as P1. After the cells reached 70-90% confluence, they were digested and cultured at 1×10 4 cell / cm 2 For subculture, the cell generation number was recorded as P2; Step S27, collect P2 cells, wash the cells with D-Hank's balanced salt solution, grind them by centrifugation and dry them to obtain the desired stem cell factors.

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