Autologous fat-derived mesenchymal stem cell composition as well as preparation method and application thereof

Abstract

The invention provides an autologous fat-derived mesenchymal stem cell composition as well as a preparation method and application thereof, and belongs to the technical field of medical auxiliary materials. The method comprises the following steps: separating mesenchymal stem cells from autologous fat particles, culturing in a stem cell culture medium added with cell stimulation particles and fetal calf serum, separating to obtain stem cells and an extracellular matrix, preparing the stem cells into stem cell lipidosome, and preparing the stem cell lipidosome into the mesenchymal stem cells. Sanguinarine and curcumin are embedded in a composite shell material of chitosan and beta-cyclodextrin to prepare the sustained release microspheres. The mesenchymal stem cell composition of the autologous fat source prepared by the invention has good air permeability, softness, certain mechanical property and good biocompatibility, can quickly promote wound healing, is convenient to remove, and does not cause secondary injury.

Description

Technical Field

[0001] The present invention relates to the technical field of medical auxiliary materials, and in particular to an autologous fat-derived mesenchymal stem cell composition, a preparation method and application thereof. Background Art

[0002] Wound healing refers to the process of tissue regeneration under a series of regulatory actions after the body has a wound due to various reasons. It includes acute wounds that can heal naturally, such as mild burns and scalds, superficial skin trauma, and ulcerative wounds that must rely on drugs and surgery. The skin wound repair process is mainly divided into several stages: coagulation period, inflammatory response period, cell growth period, and tissue regeneration. Wound repair requires the cooperation of multiple cells. The absence or delay of any link in the whole process will lead to the inability of the skin wound healing process to proceed normally. At present, the basic schemes for the treatment of skin wounds are surgery and drug therapy. Surgery generally sutures the wound surface to eventually repair the skin defect. It is a commonly used method in clinical practice and has a fast effect. However, when the wound area is large and the wound is deep, surgical debridement is likely to aggravate the condition, and for drug treatment, the usual treatment effect is mostly unsatisfactory.

[0003] Human adipose derived stem cells (ADSCs) are a type of stem cell population in the adipose matrix. They are mesenchymal stem cells (MSCs) that can self-renew and have multi-directional differentiation potential. They can differentiate into osteoblasts, chondrocytes and adipocytes in vitro. They are multipotent cells. A large number of experiments have confirmed that in a certain induction environment, adipocytes can differentiate into osteoblasts, adipocytes, chondrocytes, nerve cells, etc. They have good replacement, repair and therapeutic effects after transplantation, and are non-tumorigenic and do not cause immune rejection with the body. Stem cells themselves have the characteristics of differentiation and proliferation. In the process of wound healing, they can participate in the cell proliferation, differentiation, migration and re-formation of extracellular matrix (ECM) related to wound healing, and play a role in accelerating wound healing in many aspects. It not only speeds up the wound healing time, but also solves the problem that skin appendages such as sweat glands cannot regenerate, so that the skin can restore its original physiological function. Adipose-derived mesenchymal stem cells are widely available and easy to extract. They have the ability to self-renew and have multidirectional differentiation potential. However, when mesenchymal stem cells are transplanted to treat tissue damage, the number of cells transplanted at a time needs to be as high as 1×10 9In order to effectively play a therapeutic role, a large number of seed cells of uniform origin are required to ensure the stability and comparability of experimental results. In addition, there is a strong immune response in xenotransplantation. Therefore, a new method is needed to avoid aging and even malignant transformation of mesenchymal stem cells caused by repeated cell passages, and to be easy to obtain, store, and not induce immune rejection after transplantation. Mesenchymal stem cells secrete a variety of bioactive factors, and it has been confirmed that the paracrine function of stem cells is the main way they exert their effects.

[0004] Traditional dressings are still widely used due to their low production cost, simple manufacturing process and convenient transportation. As people's requirements for treatment gradually increase, these traditional dressings have a series of significant shortcomings, such as excessive wound tissue fluid, which increases the chance of the dressing sticking to the wound, making it easy to cause secondary damage when changing the dressing; and they cannot meet the needs of adapting to a variety of wound types; lack of controllable wound moisture absorption, resulting in excessive dryness of the wound, and inability to heal the wound in time. At present, traditional dressings are only suitable for the treatment of mild extrusion wounds or as secondary dressings.

[0005] Chinese invention patent CN116687837B discloses a stem cell preparation for promoting wound healing and its preparation method, 18.75mL of carbomer gel is mixed with 6.25mL of concentrated supernatant of human adipose mesenchymal stem cells, and then 1562.5μL of betaine working solution is added and shaken until mixed, to obtain a stem cell preparation for promoting wound healing, and the stem cell preparation is stored at 4°C. However, the stem cell preparation obtained by this patented technology has limited repair effect on wound healing, which limits its application. Summary of the invention

[0006] The purpose of the present invention is to provide an autologous fat-derived mesenchymal stem cell composition and its preparation method and application. The preparation method is simple, has good air permeability and softness, has certain mechanical properties and good biocompatibility, quickly promotes wound healing, is easy to remove, has certain hemostasis and good antibacterial properties, avoids wound infection, and will not cause secondary damage.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a method for preparing a mesenchymal stem cell composition derived from autologous fat. The mesenchymal stem cells are separated from autologous fat particles, cultured in a stem cell culture medium added with cell stimulating particles and fetal bovine serum, stem cells and extracellular matrix are separated, the stem cells are prepared into stem cell liposomes, and sanguinarine and curcumin are embedded in a composite shell material of chitosan and beta-cyclodextrin to prepare sustained-release microspheres.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Preparation of stem cell culture medium: adding cell stimulating particles and fetal bovine serum to α-MEM culture medium, and ultrasonically dispersing them uniformly to prepare stem cell culture medium;

[0011] S2. Isolation and in vitro proliferation culture of stem cells: centrifuge the autologous fat particles, collect the upper layer of fat tissue, add complex enzyme digestion, centrifuge, collect the lower layer of cells, add the stem cell culture medium in step S1 to resuspend, culture under external magnetic field conditions, centrifuge, the lower layer of the centrifugation is the autologous fat-derived mesenchymal stem cells, and the upper layer is the extracellular matrix, which is retained;

[0012] S3. Preparation of stem cell liposomes: resuspend the autologous adipose-derived mesenchymal stem cells in step S2 in a PBS solution to obtain a cell suspension; dissolve lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol in chloroform, remove the solvent by rotary evaporation under reduced pressure to obtain liposomes, add the cell suspension and mix well, and repeatedly extrude with a liposome extruder with a polycarbonate membrane to obtain stem cell liposomes;

[0013] S4. Preparation of sustained-release microspheres: chitosan was dissolved in an acid solution, β-cyclodextrin, sanguinarine and curcumin were added, an emulsifier octanol solution was added, stirred, sodium tripolyphosphate aqueous solution was added dropwise for cross-linking, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres;

[0014] S5. Preparation of an autologous fat-derived mesenchymal stem cell composition: Add N-isopropylacrylamide to the extracellular matrix in step S2, add an initiator and 2-mercaptoethylamine under inert gas protection, stir and react in an ice-water bath, add chitosan solution, EDC and NHS, stir and react at room temperature, then add silk fibroin peptide, EDC and NHS, continue to stir and react, then add stem cell liposomes and sustained-release microspheres, stir and mix evenly, and obtain an autologous fat-derived mesenchymal stem cell composition.

[0015] As a further improvement of the present invention, the concentration of the cell stimulating particles in step S1 is 2-4wt%, the concentration of the fetal bovine serum is 8-12wt%, and the preparation method of the cell stimulating particles is as follows:

[0016] T1. Preparation of UiO-66-NH2: Add zirconium chloride and 2-aminoterephthalic acid to a mixed solvent of N,N-dimethylformamide and acetic acid, perform hydrothermal reaction, cool, centrifuge, collect solids, wash, and dry to obtain UiO-66-NH2;

[0017] T2. Deposition of magnetic ferroferric oxide: UiO-66-NH2 was added to water, ferric chloride and ferrous chloride were added, ammonia water was added dropwise, heated and stirred for reaction, centrifuged, washed, dried, and calcined to obtain Fe3O4@UiO-66-NH2;

[0018] T3. Loading TGF-β1 and TGF-β3: Add TGF-β1 and TGF-β3 into water, add NHS and EDC, stir to activate, add Fe3O4@UiO-66-NH2, stir to react, centrifuge, wash, dry, and obtain cell stimulating particles.

[0019] As a further improvement of the present invention, the mass ratio of zirconium chloride and 2-aminoterephthalic acid in step T1 is 13-17:11-15, the temperature of the hydrothermal reaction is 120-140°C, and the time is 20-24h; the mass ratio of UiO-66-NH2, ferric chloride and ferrous chloride in step T2 is 10-15:3.24:1.26, the pH value of the solution is adjusted to 10-11 by dropping ammonia water, the temperature of the heating and stirring reaction is 80-90°C, the time is 3-5h, the temperature of the calcination is 550-650°C, and the time is 1-2h; the mass ratio of TGF-β1, TGF-β3, NHS, EDC and Fe3O4@UiO-66-NH2 in step T3 is 0.2-0.4:0.1-0.3:0.05-0.1:0.05-0.1:5-7.

[0020] As a further improvement of the present invention, the compound enzyme in step S2 is a mixture of trypsin and collagenase, the mass ratio is 2-4:1-3, the amount of the compound enzyme added is 1-3wt% of the mass of the upper fat tissue, the digestion temperature is 35-38°C, the time is 20-40min; the cell density after resuspension is 10 3 -10 4 / mL, the culture conditions are 35-38°C, the volume concentration of carbon dioxide is 3-7%, the time is 3-5d, and the intensity of the external magnetic field is 0.01-0.05T.

[0021] As a further improvement of the present invention, the cell density of the cell suspension in step S3 is 10 4 -10 5 g / mL, the mass ratio of lecithin, DSPE-PEG2000, DSPE-PEG-CREKA and cholesterol is 12-15:4-7:3-5:4-6, and the number of repeated extrusions is 20-30 times.

[0022] As a further improvement of the present invention, the acid solution in step S4 is a 1-3wt% acetic acid solution, the mass ratio of the chitosan, β-cyclodextrin, sanguinarine and curcumin, the emulsifier and sodium tripolyphosphate is 5-7:3-5:1-2:2-3:0.5-1:0.1-0.2, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

[0023] As a further improvement of the present invention, the initiator in step S5 is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate, and the mass ratio of N-isopropylacrylamide, initiator, 2-mercaptoethylamine, chitosan, silk fibroin peptide, stem cell liposomes and sustained-release microspheres is 18-22:3-4:10-12:10-14:7-10:8-12:5-7.

[0024] The present invention further protects an autologous fat-derived mesenchymal stem cell composition prepared by the above preparation method.

[0025] The present invention further protects the use of the above autologous fat-derived mesenchymal stem cell composition in the preparation of a drug for promoting skin healing.

[0026] The present invention has the following beneficial effects:

[0027] The present invention obtains fat-derived mesenchymal stem cells from autologous tissue, which have low immunogenicity and will not cause rejection reactions. The raw material sources are wide and the preparation method is simple. The prepared stem cell culture medium is added to carry out in vitro expansion culture. Under the action of cell stimulating particles in the culture medium and the action of an external magnetic field, the proliferation of stem cells is promoted. TGF-β1 and TGF-β3, TGF-β1 is a multifunctional cytokine that regulates the proliferation, differentiation and extracellular matrix synthesis of stem cells through the Smad signaling pathway; TGF-β3 activates the Smad pathway in cells through the signaling pathway mediated by integrins αvβ6 and αvβ8, promotes cell migration and tissue repair, and the two cooperate to regulate cell proliferation, differentiation and extracellular matrix synthesis, and play an important promoting role.

[0028] The cell stimulating particles prepared by the present invention use UiO-66-NH2 material as a carrier. -NH2 can promote the in-situ deposition of magnetic ferroferric oxide through hydrogen bonds. Ferroferric oxide not only has antibacterial properties and improves the resistance of stem cells, but also can promote the growth of stem cells by regulating cell proliferation and migration. UiO-66-NH2 has high porosity and good biocompatibility, can provide a stable microenvironment for stem cells, promote cell adhesion and growth, and carry cytokines to further promote the growth and survival of stem cells.

[0029] The present invention uses the extracellular matrix after culturing stem cells in the culture medium as a solvent to prepare the hydrogel, and the cell stimulating particles therein also have a significant effect on promoting skin healing. Among them, ferroferric oxide can also regulate cell behavior to promote scar-free healing, regulate the proliferation and migration of fibroblasts, reduce fibrosis, can effectively promote wound healing, and reduce inflammatory reactions, promote skin healing, and can also induce the regeneration of hair follicles and blood vessels. TGF-β1 can promote the transformation of fibroblasts into myofibroblasts, promote the healing process of wounds, but also lead to the formation of scars. TGF-β3 can promote the synthesis of collagen and the proliferation of fibroblasts. The ratio of TGF-β1 / TGF-β3 generation is reduced, which reduces the generation of scar tissue and achieves higher quality healing.

[0030] In addition, the active components in the extracellular matrix (exosomes, exosomes, etc.) play a vital role in regulating the conduction of cell signals around the wound. The hydrogel scaffold provides physical isolation for the wound surface and promotes cell adhesion around the wound tissue. At the same time, the addition of hydrogel can slow down the release of active components. The active components continuously released at the wound site can better stimulate wound healing, while acting as a "sacrificial substrate" in the wound, delaying the erosion of wound tissue by multiple enzymes at the wound site. The combination of the two can accelerate wound healing by regulating the wound microenvironment, promoting collagen synthesis, cell migration and angiogenesis.

[0031] The hydrogel of the present invention uses poly N-isopropylacrylamide (PNIPAAM) as one of the polymer materials. Since the lower critical solution temperature (about 32°C) of its aqueous solution is close to the normal temperature of the human body (37°C), when the temperature is higher than 32°C, the hydrogen bonds in PNIPAAM itself show contraction, thereby promoting the release of the loaded drug and realizing the controlled release of the drug. The modified scaffold structure formed by chitosan and silk protein peptide greatly improves the antibacterial property, low sensitization and high biocompatibility of the hydrogel. After the complex hydrogel system is formed through temperature changes and a solid hydrogel system is formed, the hydrogen bonds show contraction, which promotes the release of stem cell liposomes and sustained-release microspheres, thereby promoting the healing of the skin.

[0032] The present invention embeds stem cells through liposomes, which improves the transdermal absorption effect of stem cells, has the effect of targeted release and efficient delivery, and can protect stem cells from the influence of the external environment, prolonging their survival time and activity in the body. Curcumin and sanguinarine are embedded in the sustained-release microspheres of the present invention, and the embedding layer includes cyclodextrin. The cavity of cyclodextrin can load the poorly soluble drug sanguinarine, thereby greatly increasing the loading amount. Sanguinarine has several antibacterial and inflammatory response reducing effects, and curcumin has a strong antioxidant effect, which can reduce the overexpression of reactive oxygen species that can lead to chronic inflammation, thereby promoting skin wound healing.

[0033] The present invention prepares a mesenchymal stem cell composition derived from autologous fat, which is a thermosensitive hydrogel and is liquid at room temperature. A layer of the composition is evenly dripped on a skin injury or damage, and is rapidly solidified to form a hydrogel layer under body temperature. The hydrogel, as a hydrophilic gel with a three-dimensional mesh structure, has good air permeability and softness, certain mechanical properties and good biocompatibility. At the same time, the hydrogel structure can maintain a relatively moist environment at the skin and quickly promote wound healing; the internal polymer structure has pores, which can enable necessary gas exchange between the damaged tissue and the environment; at the same time, the hydrogel material can absorb cell tissue fluid, has certain hemostasis and good antibacterial properties, and avoids wound infection; it is non-toxic and easy to prepare; it has suitable adhesion to wound tissue, is easy to remove, and will not cause secondary damage. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0035] NHS, N-hydroxysuccinimide, EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, PBS, phosphate buffered saline, TGF-β1, transforming growth factor-β1; TGF-β3, transforming growth factor-β3, DSPE-PEG2000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, DSPE-PEG-CREKA, distearoylphosphatidylethanolamine-polyethylene glycol-tumor targeting protein. Trypsin, 2500U / mg, collagenase 125U / mg. α-MEM culture medium, Merck Biotechnology.

[0036] Preparation Example 1 Preparation of cell stimulating particles

[0037] Here’s how:

[0038] T1. Preparation of UiO-66-NH2: 1.3 g zirconium chloride and 1.1 g 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio of 24:1), and hydrothermally reacted at 120 °C for 20 h. The mixture was cooled, centrifuged, and the solid was collected, washed, and dried to obtain UiO-66-NH2.

[0039] T2. Deposition of magnetic ferroferric oxide: 10 g UiO-66-NH2 was added to 200 mL water, 3.24 g ferric chloride and 1.26 g ferrous chloride were added, ammonia water was added to adjust the pH value of the solution to 10, heated to 80 ° C, stirred for 3 h, centrifuged, washed, dried, and calcined at 550 ° C for 1 h to obtain Fe3O4@UiO-66-NH2;

[0040] T3. Loading TGF-β1 and TGF-β3: Add 0.2g TGF-β1 and 0.1g TGF-β3 into 100mL water, add 0.05g NHS and 0.05g EDC, stir and activate for 30min, add 5g Fe3O4@UiO-66-NH2, stir and react for 8h, centrifuge, wash, and dry to obtain cell stimulating particles.

[0041] Preparation Example 2 Preparation of cell stimulating particles

[0042] Here’s how:

[0043] T1. Preparation of UiO-66-NH2: 1.7 g zirconium chloride and 1.5 g 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio of 24:1), and hydrothermally reacted at 140 °C for 24 h. The mixture was cooled, centrifuged, and the solid was collected, washed, and dried to obtain UiO-66-NH2.

[0044] T2. Deposition of magnetic ferroferric oxide: 15 g UiO-66-NH2 was added to 200 mL water, 3.24 g ferric chloride and 1.26 g ferrous chloride were added, ammonia water was added to adjust the pH value of the solution to 11, heated to 90 ° C, stirred for 5 h, centrifuged, washed, dried, and calcined at 650 ° C for 2 h to obtain Fe3O4@UiO-66-NH2;

[0045] T3. Loading TGF-β1 and TGF-β3: Add 0.4g TGF-β1 and 0.3g TGF-β3 into 100mL water, add 0.1g NHS and 0.1g EDC, stir and activate for 30min, add 7g Fe3O4@UiO-66-NH2, stir and react for 10h, centrifuge, wash, and dry to obtain cell stimulating particles.

[0046] Preparation Example 3 Preparation of cell stimulating particles

[0047] Here’s how:

[0048] T1. Preparation of UiO-66-NH2: 1.5 g zirconium chloride and 1.3 g 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio of 24:1), and hydrothermally reacted at 130 °C for 22 h. The mixture was cooled, centrifuged, and the solid was collected, washed, and dried to obtain UiO-66-NH2.

[0049] T2. Deposition of magnetic ferroferric oxide: 12 g UiO-66-NH2 was added to 200 mL water, 3.24 g ferric chloride and 1.26 g ferrous chloride were added, ammonia water was added to adjust the solution pH to 10.5, heated to 85 °C, stirred for 4 h, centrifuged, washed, dried, and calcined at 600 °C for 1.5 h to obtain Fe3O4@UiO-66-NH2;

[0050] T3. Loading TGF-β1 and TGF-β3: Add 0.3g TGF-β1 and 0.2g TGF-β3 into 100mL water, add 0.07g NHS and 0.07g EDC, stir and activate for 30min, add 6g Fe3O4@UiO-66-NH2, stir and react for 9h, centrifuge, wash, and dry to obtain cell stimulating particles.

[0051] Comparative Preparation Example 1

[0052] Compared with Preparation Example 3, the difference is that step T2 is not performed.

[0053] The details are as follows:

[0054] T1. Preparation of UiO-66-NH2: 1.5 g zirconium chloride and 1.3 g 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio of 24:1), and hydrothermally reacted at 130 °C for 22 h. The mixture was cooled, centrifuged, and the solid was collected, washed, and dried to obtain UiO-66-NH2.

[0055] T2. Loading TGF-β1 and TGF-β3: Add 0.3g TGF-β1 and 0.2g TGF-β3 into 100mL water, add 0.07g NHS and 0.07g EDC, stir and activate for 30min, add 6g UiO-66-NH2, stir and react for 9h, centrifuge, wash, and dry to obtain cell stimulating particles.

[0056] Comparative Preparation Example 2

[0057] The difference compared with Preparation Example 3 is that TGF-β1 is not added in step T3.

[0058] The details are as follows:

[0059] T3. Loading TGF-β3: Add 0.5 g TGF-β3 into 100 mL water, add 0.07 g NHS and 0.07 g EDC, stir and activate for 30 min, add 6 g Fe3O4@UiO-66-NH2, stir and react for 9 h, centrifuge, wash, and dry to obtain cell stimulating particles.

[0060] Comparative Preparation Example 3

[0061] Compared with Preparation Example 3, the difference is that TGF-β3 is not added in step T3.

[0062] The details are as follows:

[0063] T3. Loading TGF-β1: Add 0.5 g TGF-β1 into 100 mL water, add 0.07 g NHS and 0.07 g EDC, stir and activate for 30 min, add 6 g Fe3O4@UiO-66-NH2, stir and react for 9 h, centrifuge, wash, and dry to obtain cell stimulating particles.

[0064] Comparative Preparation Example 4

[0065] Compared with Preparation Example 3, the difference is that step T3 is not performed.

[0066] The details are as follows:

[0067] T1. Preparation of UiO-66-NH2: 1.5 g zirconium chloride and 1.3 g 2-aminoterephthalic acid were added to 200 mL of a mixed solvent of N,N-dimethylformamide and acetic acid (volume ratio of 24:1), and hydrothermally reacted at 130 °C for 22 h. The mixture was cooled, centrifuged, and the solid was collected, washed, and dried to obtain UiO-66-NH2.

[0068] T2. Deposition of magnetic ferroferric oxide: 12 g UiO-66-NH2 was added to 200 mL water, 3.24 g ferric chloride and 1.26 g ferrous chloride were added, ammonia was added dropwise to adjust the pH value of the solution to 10.5, heated to 85 °C, stirred for 4 h, centrifuged, washed, dried, and calcined at 600 °C for 1.5 h to obtain Fe3O4@UiO-66-NH2, which is the cell stimulating particle. 。

[0069] Example 1

[0070] This embodiment provides a method for preparing an autologous fat-derived mesenchymal stem cell composition, comprising the following steps:

[0071] S1. Preparation of stem cell culture medium: adding the cell stimulating particles prepared in Preparation Example 1 and fetal bovine serum to the α-MEM culture medium, and ultrasonically dispersing at 1000W for 15min to prepare a stem cell culture medium, wherein the concentration of the cell stimulating particles is 2wt%, and the concentration of the fetal bovine serum is 8wt%;

[0072] S2. Isolation and in vitro proliferation culture of stem cells: Take the fat particles extracted from the body, centrifuge at 1000r / min for 5min, collect the upper layer of fat tissue from this centrifugation, add the complex enzyme, the amount of the complex enzyme added is 1wt% of the mass of the upper layer of fat tissue, digest at 35℃ for 20min, centrifuge at 700g / min for 5min, collect the lower layer of cells from this centrifugation, add the stem cell culture medium in step S1 to resuspend, and the cell density is 10 3 -10 4 / mL, under the conditions of 0.01T external magnetic field, 35℃, 3% carbon dioxide volume concentration, culture for 3 days, and centrifuge at 700g / min for 10min. The lower layer of this centrifugation is autologous fat-derived mesenchymal stem cells, and the upper layer is extracellular matrix, which is retained;

[0073] The complex enzyme is a mixture of trypsin and collagenase, with a mass ratio of 2:1;

[0074] S3. Preparation of stem cell liposomes: The autologous fat-derived mesenchymal stem cells in step S2 were resuspended in PBS solution to obtain a cell suspension with a cell density of 10 4 -10 5 g / mL; 1.2g lecithin, 0.4g DSPE-PEG2000, 0.3g DSPE-PEG-CREKA, and 0.4g cholesterol were dissolved in 50mL chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, 50mL of cell suspension was added and mixed evenly, and the mixture was repeatedly extruded 20 times using a liposome extruder with a polycarbonate membrane to prepare stem cell liposomes;

[0075] S4. Preparation of sustained-release microspheres: 5 g of chitosan was dissolved in 200 mL of 1 wt% acetic acid solution, 3 g of β-cyclodextrin, 1 g of sanguinarine and 2 g of curcumin were added, 50 mL of octanol solution containing 0.5 g of Tween-20 was added, stirred for 30 min, 10 mL of an aqueous solution containing 0.1 g of sodium tripolyphosphate was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres;

[0076] S5. Preparation of autologous adipose-derived mesenchymal stem cell composition: 1.8 g of N-isopropylacrylamide was added to 200 mL of the extracellular matrix in step S2, and 0.3 g of potassium persulfate and 1 g of 2-mercaptoethylamine were added under nitrogen protection, and the mixture was stirred for reaction in an ice-water bath for 3 h, and 30 mL of acetic acid solution containing 1 g of chitosan (2 wt% of acetic acid content), 0.3 g of EDC and 0.2 g of NHS were added, and the mixture was stirred for reaction at room temperature for 20 h, and then 0.7 g of silk fibroin peptide, 0.2 g of EDC and 0.1 g of NHS were added, and the mixture was stirred for reaction for 20 h, and then 0.8 g of stem cell liposomes and 0.5 g of sustained-release microspheres were added, and the mixture was stirred for 30 min to obtain an autologous adipose-derived mesenchymal stem cell composition.

[0077] Example 2

[0078] This embodiment provides a method for preparing an autologous fat-derived mesenchymal stem cell composition, comprising the following steps:

[0079] S1. Preparation of stem cell culture medium: adding the cell stimulating particles prepared in Preparation Example 2 and fetal bovine serum to the α-MEM culture medium, and ultrasonically dispersing at 1000W for 15min to prepare a stem cell culture medium, wherein the concentration of the cell stimulating particles is 4wt%, and the concentration of the fetal bovine serum is 12wt%;

[0080] S2. Isolation and in vitro proliferation culture of stem cells: Take the fat particles extracted from the body, centrifuge at 1000r / min for 5min, collect the upper layer of fat tissue from this centrifugation, add the complex enzyme, the amount of the complex enzyme added is 3wt% of the mass of the upper layer of fat tissue, digest at 38℃ for 40min, centrifuge at 700g / min for 5min, collect the lower layer of cells from this centrifugation, add the stem cell culture medium in step S1 to resuspend, and the cell density is 10 3 -10 4 / mL, under the conditions of 0.05T external magnetic field, 38℃, 7% carbon dioxide volume concentration, culture for 5 days, and centrifuge at 700g / min for 10min. The lower layer of this centrifugation is autologous fat-derived mesenchymal stem cells, and the upper layer is extracellular matrix, which is retained;

[0081] The complex enzyme is a mixture of trypsin and collagenase, with a mass ratio of 4:3;

[0082] S3. Preparation of stem cell liposomes: The autologous fat-derived mesenchymal stem cells in step S2 were resuspended in PBS solution to obtain a cell suspension with a cell density of 10 4 -10 5g / mL; 1.5 g of lecithin, 0.7 g of DSPE-PEG2000, 0.5 g of DSPE-PEG-CREKA, and 0.6 g of cholesterol were dissolved in 50 mL of chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, 50 mL of cell suspension was added and mixed evenly, and the mixture was repeatedly extruded 30 times using a liposome extruder with a polycarbonate membrane to prepare stem cell liposomes;

[0083] S4. Preparation of sustained-release microspheres: 7 g of chitosan was dissolved in 200 mL of 3 wt% acetic acid solution, 5 g of β-cyclodextrin, 2 g of sanguinarine and 3 g of curcumin were added, 50 mL of octanol solution containing 1 g of Tween-60 was added, stirred for 30 min, 10 mL of an aqueous solution containing 0.2 g of sodium tripolyphosphate was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres;

[0084] S5. Preparation of autologous adipose-derived mesenchymal stem cell composition: 2.2 g of N-isopropylacrylamide was added to 200 mL of the extracellular matrix in step S2, and 0.4 g of sodium persulfate and 1.2 g of 2-mercaptoethylamine were added under nitrogen protection, and the mixture was stirred for reaction in an ice-water bath for 5 h, and 30 mL of acetic acid solution containing 1.4 g of chitosan (2 wt% of acetic acid content), 0.5 g of EDC and 0.3 g of NHS were added, and the mixture was stirred for reaction at room temperature for 24 h, and then 1 g of silk fibroin peptide, 0.4 g of EDC and 0.2 g of NHS were added, and the mixture was stirred for reaction for 24 h, and then 1.2 g of stem cell liposomes and 0.7 g of sustained-release microspheres were added, and the mixture was stirred for 30 min to obtain an autologous adipose-derived mesenchymal stem cell composition.

[0085] Example 3

[0086] This embodiment provides a method for preparing an autologous fat-derived mesenchymal stem cell composition, comprising the following steps:

[0087] S1. Preparation of stem cell culture medium: adding the cell stimulating particles prepared in Preparation Example 3 and fetal bovine serum to the α-MEM culture medium, and ultrasonically dispersing at 1000W for 15min to prepare a stem cell culture medium, wherein the concentration of the cell stimulating particles is 3wt%, and the concentration of the fetal bovine serum is 10wt%;

[0088] S2. Isolation and in vitro proliferation culture of stem cells: Take the fat particles extracted from the body, centrifuge at 1000r / min for 5min, collect the upper layer of fat tissue from this centrifugation, add the complex enzyme, the amount of the complex enzyme added is 2wt% of the mass of the upper layer of fat tissue, digest at 37℃ for 30min, centrifuge at 700g / min for 5min, collect the lower layer of cells from this centrifugation, add the stem cell culture medium in step S1 to resuspend, and the cell density is 10 3 -10 4 / mL, under the conditions of 0.03T external magnetic field, 37℃, 5% carbon dioxide volume concentration, culture for 4 days, and centrifuge at 700g / min for 10min. The lower layer of this centrifugation is autologous fat-derived mesenchymal stem cells, and the upper layer is extracellular matrix, which is retained;

[0089] The complex enzyme is a mixture of trypsin and collagenase, with a mass ratio of 3:2;

[0090] S3. Preparation of stem cell liposomes: The autologous fat-derived mesenchymal stem cells in step S2 were resuspended in PBS solution to obtain a cell suspension with a cell density of 10 4 -10 5 g / mL; 1.3g lecithin, 0.55g DSPE-PEG2000, 0.4g DSPE-PEG-CREKA, and 0.5g cholesterol were dissolved in 50mL chloroform, and the solvent was removed by rotary evaporation under reduced pressure to prepare liposomes, 50mL of cell suspension was added and mixed evenly, and the mixture was repeatedly extruded 25 times using a liposome extruder with a polycarbonate membrane to prepare stem cell liposomes;

[0091] S4. Preparation of sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 2 wt% acetic acid solution, 4 g of β-cyclodextrin, 1.5 g of sanguinarine and 2.5 g of curcumin were added, 50 mL of octanol solution containing 0.7 g of Tween-85 was added, stirred for 30 min, 10 mL of an aqueous solution containing 0.15 g of sodium tripolyphosphate was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres;

[0092] S5. Preparation of autologous adipose-derived mesenchymal stem cell composition: 2 g of N-isopropylacrylamide was added to 200 mL of the extracellular matrix in step S2, and 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine were added under nitrogen protection, and the mixture was stirred for reaction in an ice-water bath for 4 h, and 30 mL of acetic acid solution containing 1.2 g of chitosan (2 wt% of acetic acid content), 0.4 g of EDC and 0.25 g of NHS were added, and the mixture was stirred for reaction at room temperature for 22 h, and then 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS were added, and the mixture was stirred for reaction for 22 h, and then 1 g of stem cell liposomes and 0.6 g of sustained-release microspheres were added, and the mixture was stirred for 30 min to obtain an autologous adipose-derived mesenchymal stem cell composition.

[0093] Comparative Example 1

[0094] Compared with Example 3, the difference is that the cell stimulating particles are prepared by Comparative Preparation Example 1.

[0095] Comparative Example 2

[0096] Compared with Example 3, the difference is that the cell stimulating particles are prepared by Comparative Preparation Example 2.

[0097] Comparative Example 3

[0098] Compared with Example 3, the difference is that the cell stimulating particles are prepared by Comparative Preparation Example 3.

[0099] Comparative Example 4

[0100] Compared with Example 3, the difference is that the cell stimulating particles are prepared by Comparative Preparation Example 4.

[0101] Comparative Example 5

[0102] The difference compared with Example 3 is that no cell stimulating particles were added.

[0103] The details are as follows:

[0104] S1. Preparation of stem cell culture medium: fetal bovine serum was added to α-MEM culture medium, and ultrasonic dispersion was performed at 1000W for 15 minutes to prepare a stem cell culture medium, wherein the concentration of the fetal bovine serum was 10wt%.

[0105] Comparative Example 6

[0106] Compared with Example 3, the difference is that step S3 is not performed.

[0107] The details are as follows:

[0108] S1. Preparation of stem cell culture medium: adding the cell stimulating particles prepared in Preparation Example 3 and fetal bovine serum to the α-MEM culture medium, and ultrasonically dispersing at 1000W for 15min to prepare a stem cell culture medium, wherein the concentration of the cell stimulating particles is 3wt%, and the concentration of the fetal bovine serum is 10wt%;

[0109] S2. Isolation and in vitro proliferation culture of stem cells: Take the fat particles extracted from the body, centrifuge at 1000r / min for 5min, collect the upper layer of fat tissue from this centrifugation, add the complex enzyme, the amount of the complex enzyme added is 2wt% of the mass of the upper layer of fat tissue, digest at 37℃ for 30min, centrifuge at 700g / min for 5min, collect the lower layer of cells from this centrifugation, add the stem cell culture medium in step S1 to resuspend, and the cell density is 10 3 -10 4 / mL, under the conditions of 0.03T external magnetic field, 37℃, 5% carbon dioxide volume concentration, culture for 4 days, and centrifuge at 700g / min for 10min. The lower layer of this centrifugation is autologous fat-derived mesenchymal stem cells, and the upper layer is extracellular matrix, which is retained;

[0110] The complex enzyme is a mixture of trypsin and collagenase, with a mass ratio of 3:2;

[0111] S3. Preparation of sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 2 wt% acetic acid solution, 4 g of β-cyclodextrin, 1.5 g of sanguinarine and 2.5 g of curcumin were added, 50 mL of octanol solution containing 0.7 g of Tween-85 was added, stirred for 30 min, 10 mL of an aqueous solution containing 0.15 g of sodium tripolyphosphate was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres;

[0112] S4. Preparation of autologous adipose-derived mesenchymal stem cell composition: 2 g of N-isopropylacrylamide was added to 200 mL of the extracellular matrix in step S2, and 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine were added under nitrogen protection, and the mixture was stirred for reaction in an ice-water bath for 4 h. 30 mL of acetic acid solution containing 1.2 g of chitosan (2 wt% of acetic acid content), 0.4 g of EDC and 0.25 g of NHS were added, and the mixture was stirred for reaction at room temperature for 22 h. Then, 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS were added, and the mixture was stirred for reaction for 22 h. Then, 1 g of the autologous adipose-derived mesenchymal stem cells in step S2 and 0.6 g of sustained-release microspheres were added, and the mixture was stirred for 30 min to obtain an autologous adipose-derived mesenchymal stem cell composition.

[0113] Comparative Example 7

[0114] Compared with Example 3, the difference is that no sanguinarine is added in step S4.

[0115] The details are as follows:

[0116] S4. Preparation of sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 2 wt% acetic acid solution, 4 g of β-cyclodextrin, 4 g of curcumin, 50 mL of octanol solution containing 0.7 g of Tween-85 were added, stirred for 30 min, 10 mL of aqueous solution containing 0.15 g of sodium tripolyphosphate was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres.

[0117] Comparative Example 8

[0118] Compared with Example 3, the difference is that curcumin is not added in step S4.

[0119] The details are as follows:

[0120] S4. Preparation of sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 2 wt% acetic acid solution, 4 g of β-cyclodextrin, 4 g of sanguinarine, and 50 mL of octanol solution containing 0.7 g of Tween-85 were added, stirred for 30 min, 10 mL of an aqueous solution containing 0.15 g of sodium tripolyphosphate was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres.

[0121] Comparative Example 9

[0122] Compared with Example 3, the difference is that no sustained-release microspheres are added in step S5.

[0123] The details are as follows:

[0124] S5. Preparation of autologous adipose-derived mesenchymal stem cell composition: 2 g of N-isopropylacrylamide was added to 200 mL of the extracellular matrix in step S2, and under nitrogen protection, 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine were added, and the mixture was stirred for 4 h in an ice-water bath, and 30 mL of acetic acid solution containing 1.2 g of chitosan (2 wt% of acetic acid content), 0.4 g of EDC and 0.25 g of NHS were added, and the mixture was stirred for 22 h at room temperature, and then 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS were added, and the mixture was stirred for 22 h, and then 1 g of stem cell liposomes was added, and the mixture was stirred for 30 min to obtain an autologous adipose-derived mesenchymal stem cell composition.

[0125] Comparative Example 10

[0126] Compared with Example 3, the difference is that no stem cell liposomes are added in step S5.

[0127] The details are as follows:

[0128] S5. Preparation of autologous adipose-derived mesenchymal stem cell composition: 2 g of N-isopropylacrylamide was added to 200 mL of the extracellular matrix in step S2, and under nitrogen protection, 0.35 g of ammonium persulfate and 1.1 g of 2-mercaptoethylamine were added, and the mixture was stirred for reaction in an ice-water bath for 4 h, and 30 mL of acetic acid solution containing 1.2 g of chitosan (2 wt% of acetic acid content), 0.4 g of EDC and 0.25 g of NHS were added, and the mixture was stirred for reaction at room temperature for 22 h, and then 0.85 g of silk fibroin peptide, 0.3 g of EDC and 0.15 g of NHS were added, and the reaction was continued with stirring for 22 h, and then 0.6 g of sustained-release microspheres were added, and the mixture was stirred and mixed for 30 min to obtain an autologous adipose-derived mesenchymal stem cell composition.

[0129] Test Example 1 Determination of gelation temperature and gelation time

[0130] The autologous fat-derived mesenchymal stem cell composition prepared in Examples 1-3 and the commercially available poly (N-isopropylacrylamide) hydrogel were placed in a glass test tube and placed in a constant temperature water bath. The test tube was taken out every 10 minutes and inverted to observe whether the hydrogel flowed. If it flowed, the temperature was continued to rise at a rate of 1°C. If it did not flow, this temperature was defined as the T of the hydrogel. gel . Determine T gel Afterwards, the gel time of each group of hydrogels was measured at 37°C, and the gel time was recorded as the time point when each group of samples stopped flowing. The results are shown in Table 1.

[0131] Table 1

[0132] It can be seen from the above table that the autologous fat-derived mesenchymal stem cell compositions prepared in Examples 1-3 of the present invention have a suitable gelation temperature and a short gelation time.

[0133] Test Example 2

[0134] After BALB / c mice were adaptively fed for 1 week, they were divided into model group, Example 1-3 group, and Comparative Example 1-10 group according to the random number table method, with 6 mice in each group. A mouse model of chronic skin ulcer was established in each group. The specific operation was as follows: the back of the mouse was shaved, and the hair was removed with 6% sodium sulfide, and 0.3% sodium pentobarbital (45 mg / kg) was injected intraperitoneally for anesthesia. A transverse incision of about 5 mm in length was selected on the back, a metal ring was buried at the incision, and the skin tissue was cut along the inner edge of the ring to form a defective wound with a diameter of about 8 mm. The metal ring was removed to complete the modeling. Blank group: no treatment was performed. Before each administration in other groups, the mice were cleaned with physiological saline and furazolidone solution. Model group: only furazolidone solution was applied externally; in Example 1-3 or Comparative Example 1-10 group, 2 mL of the corresponding drug was dripped onto the wound surface so that it could be evenly covered on the wound surface and form a hydrogel. Once a day for 14 consecutive days.

[0135] On the 1st and 14th days of administration, the wound area was recorded using Image Pro Plus 6.0 software, and the wound healing rate was calculated. Wound healing rate (%) = 1-[(wound area on the first day - wound area on the day of administration) / wound area on the first day] × 100%.

[0136] The results are shown in Table 2.

[0137] Table 2

[0138] Notes: Compared with the model group, P<0.05.

[0139] It can be seen from the above table that the autologous fat-derived mesenchymal stem cell compositions prepared in Examples 1-3 of the present invention have a good effect of promoting skin healing.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an autologous fat-derived mesenchymal stem cell composition, characterized in that: Mesenchymal stem cells were separated from autologous fat particles and cultured in a stem cell culture medium added with cell stimulating particles and fetal bovine serum to separate stem cells and extracellular matrix. The stem cells were prepared into stem cell liposomes, and sanguinarine and curcumin were embedded in a composite shell material of chitosan and β-cyclodextrin to prepare sustained-release microspheres.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Preparation of stem cell culture medium: adding cell stimulating particles and fetal bovine serum to α-MEM culture medium, and ultrasonically dispersing them uniformly to prepare stem cell culture medium; S2. Isolation and in vitro proliferation culture of stem cells: centrifuge the autologous fat particles, collect the upper layer of fat tissue, add complex enzyme digestion, centrifuge, collect the lower layer of cells, add the stem cell culture medium in step S1 to resuspend, culture under external magnetic field conditions, centrifuge, the lower layer of the centrifugation is the autologous fat-derived mesenchymal stem cells, and the upper layer is the extracellular matrix, which is retained; S3. Preparation of stem cell liposomes: resuspend the autologous adipose-derived mesenchymal stem cells in step S2 in a PBS solution to obtain a cell suspension; dissolve lecithin, DSPE-PEG2000, DSPE-PEG-CREKA, and cholesterol in chloroform, remove the solvent by rotary evaporation under reduced pressure to obtain liposomes, add the cell suspension and mix well, and repeatedly extrude with a liposome extruder with a polycarbonate membrane to obtain stem cell liposomes; S4. Preparation of sustained-release microspheres: chitosan was dissolved in an acid solution, β-cyclodextrin, sanguinarine and curcumin were added, an emulsifier octanol solution was added, stirred, sodium tripolyphosphate aqueous solution was added dropwise for cross-linking, centrifuged, washed, and freeze-dried to obtain sustained-release microspheres; S5. Preparation of an autologous fat-derived mesenchymal stem cell composition: Add N-isopropylacrylamide to the extracellular matrix in step S2, add an initiator and 2-mercaptoethylamine under inert gas protection, stir and react in an ice-water bath, add chitosan solution, EDC and NHS, stir and react at room temperature, then add silk fibroin peptide, EDC and NHS, continue to stir and react, then add stem cell liposomes and sustained-release microspheres, stir and mix evenly, and obtain an autologous fat-derived mesenchymal stem cell composition.

3. The preparation method according to claim 2, characterized in that: In step S1, the concentration of the cell stimulating particles is 2-4wt%, the concentration of the fetal bovine serum is 8-12wt%, and the preparation method of the cell stimulating particles is as follows: T1. Preparation of UiO-66-NH2: Add zirconium chloride and 2-aminoterephthalic acid to a mixed solvent of N,N-dimethylformamide and acetic acid, perform hydrothermal reaction, cool, centrifuge, collect solids, wash, and dry to obtain UiO-66-NH2; T2. Deposition of magnetic ferroferric oxide: UiO-66-NH2 was added to water, ferric chloride and ferrous chloride were added, ammonia water was added dropwise, heated and stirred for reaction, centrifuged, washed, dried, and calcined to obtain Fe3O4@UiO-66-NH2; T3. Loading TGF-β1 and TGF-β3: Add TGF-β1 and TGF-β3 into water, add NHS and EDC, stir to activate, add Fe3O4@UiO-66-NH2, stir to react, centrifuge, wash, dry, and obtain cell stimulating particles.

4. The preparation method according to claim 3, characterized in that: The mass ratio of zirconium chloride and 2-aminoterephthalic acid in step T1 is 13-17:11-15, the temperature of the hydrothermal reaction is 120-140°C, and the time is 20-24h; the mass ratio of UiO-66-NH2, ferric chloride and ferrous chloride in step T2 is 10-15: 3.24:1.26, the pH value of the solution is adjusted to 10-11 by dropping ammonia water, the temperature of the heating and stirring reaction is 80-90°C, the time is 3-5h, the temperature of the calcination is 550-650°C, and the time is 1-2h; the mass ratio of TGF-β1, TGF-β3, NHS, EDC and Fe3O4@UiO-66-NH2 in step T3 is 0.2-0.4: 0.1-0.3: 0.05-0.1: 0.05-0.1: 5-7.

5. The preparation method according to claim 2, characterized in that: In step S2, the compound enzyme is a mixture of trypsin and collagenase, with a mass ratio of 2-4:1-3, and the amount of the compound enzyme added is 1-3wt% of the mass of the upper fat tissue. The digestion temperature is 35-38°C and the time is 20-40min. The cell density after resuspension is 10 3 -10 4 / mL, the culture conditions are 35-38°C, the volume concentration of carbon dioxide is 3-7%, the time is 3-5d, and the intensity of the external magnetic field is 0.01-0.05T.

6. The preparation method according to claim 2, characterized in that: The cell density of the cell suspension in step S3 is 10 4 -10 5 g / mL, the mass ratio of lecithin, DSPE-PEG2000, DSPE-PEG-CREKA and cholesterol is 12-15:4-7:3-5:4-6, and the number of repeated extrusions is 20-30 times.

7. The preparation method according to claim 2, characterized in that: The acid solution in step S4 is a 1-3wt% acetic acid solution, the mass ratio of the chitosan, β-cyclodextrin, sanguinarine and curcumin, the emulsifier and sodium tripolyphosphate is 5-7:3-5:1-2:2-3:0.5-1:0.1-0.2, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

8. The preparation method according to claim 2, characterized in that: In step S5, the initiator is selected from at least one of sodium persulfate, potassium persulfate, and ammonium persulfate, and the mass ratio of N-isopropylacrylamide, initiator, 2-mercaptoethylamine, chitosan, silk fibroin peptide, stem cell liposome and sustained-release microspheres is 18-22:3-4:10-12:10-14:7-10:8-12:5-7.

9. An autologous fat-derived mesenchymal stem cell composition obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the autologous fat-derived mesenchymal stem cell composition according to claim 9 in the preparation of a drug for promoting skin healing.

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