Stem cell exosome preparation for anti-aging repair and preparation method thereof

Abstract

The invention provides a stem cell exosome preparation for anti-aging repair and a preparation method thereof, and belongs to the technical field of skin medicine. Every 100 g of the preparation comprises the following components: 0.002 g of adipose tissue-derived stromal cell exosome, 0.008 g of volvariella volvacea polysaccharide, 0.2 g of sodium hyaluronate, 3 g of glycerin, 0.8 g of carbomer, 0.5 g of phenoxyethanol, and the balance of distilled water, totaling 100 g. Wherein the volvariella volvacea polysaccharide is prepared through specific alkali extraction and dialysis purification methods, so that the volvariella volvacea polysaccharide has special biological activity. According to the preparation, the stem cell exosome and the straw mushroom polysaccharide are combined according to the mass ratio of 1: 4, the synergistic effect is achieved, the survival rate and the oxidation resistance of skin cells are remarkably improved, and skin photoaging damage caused by ultraviolet rays is effectively prevented. Experimental results show that compared with single use of the exosome or the straw mushroom polysaccharide, the preparation has better effects in the aspects of improving the SOD level and reducing the MDA content.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin medicine, and in particular relates to a stem cell exosome preparation for anti-aging and repair. Background Art

[0002] Skin aging is the most intuitive manifestation of human aging. As we age, our skin gradually loses its elasticity and luster and develops wrinkles, sagging, pigmentation, etc. The skin aging process is affected by a variety of internal and external factors. Internal factors include decreased cell function, reduced collagen and elastic fibers, and weakened cell proliferation, while external factors are mainly the effects of ultraviolet rays, pollutants, oxidative stress, and chronic inflammation.

[0003] In recent years, exosomes, as an emerging anti-aging strategy, have attracted widespread attention due to their unique functions in intercellular signaling. Exosomes are nanoscale vesicles secreted by cells that can deliver a variety of active molecules such as proteins, lipids, mRNA and miRNA, and regulate the physiological functions of receptor cells. Exosomes derived from stem cells, especially mesenchymal stem cell exosomes, have been found to delay skin aging by promoting collagen production, enhancing skin barrier repair, and reducing the release of inflammatory factors. In addition, exosomes can repair skin damage caused by ultraviolet rays or pollution, restore the skin's health, and show good anti-aging potential.

[0004] However, although exosomes have certain tissue repair and anti-aging effects, a single exosome component alone cannot fully cope with the various biological mechanisms involved in the aging process. The oxidative stress, inflammatory response, genetic damage, cell regeneration disorders, etc. involved in the aging process are complex and multi-layered, and a single pathway of action of exosomes may not be sufficient to achieve significant anti-aging effects. Secondly, the extraction and purification process of exosomes is relatively complicated, and the high cost limits its promotion in large-scale commercial applications. Therefore, further improving the application effect of exosomes in anti-aging by combining other active ingredients is a problem that needs to be solved at present. Summary of the invention

[0005] The purpose of the present invention is to provide a stem cell exosome preparation for anti-aging and repair and a preparation method thereof, thereby effectively improving the anti-aging effect of exosomes.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] First, the present invention provides a stem cell exosome preparation for anti-aging and repair, wherein each 100 g of the stem cell exosome preparation comprises the following components:

[0008] Stem cell exosomes: 0.002g,

[0009] Volvariella polysaccharide: 0.008g,

[0010] Sodium hyaluronate: 0.2g,

[0011] Glycerin: 3g,

[0012] Carbomer: 0.8g,

[0013] Phenoxyethanol: 0.5g,

[0014] Distilled water: up to 100g.

[0015] Preferably, the stem cell exosomes are adipose-derived mesenchymal stem cell exosomes;

[0016] The straw mushroom polysaccharide is prepared by the following preparation method:

[0017] (1) freeze-drying the straw mushrooms in a vacuum oven and then grinding them into fine powder to obtain straw mushroom fine powder A;

[0018] (2) refluxing the straw mushroom powder with ethanol to remove lipid substances;

[0019] (3) After filtering, vacuum freeze drying is performed to obtain straw mushroom fine powder B;

[0020] (4) adding the straw mushroom powder B into a 0.2 M NaOH solution at a solid-liquid ratio of 1:20, heating to 50° C., and performing alkaline extraction for 3 hours with continuous stirring;

[0021] (5) After adjusting the pH of the extract to neutral, centrifuge and collect the supernatant;

[0022] (6) Add 4 times the volume of anhydrous ethanol to the supernatant and let it stand for 12 hours;

[0023] (7) collecting the precipitate by centrifugation, drying it, and obtaining crude polysaccharide of the straw mushroom, and preparing the crude polysaccharide of the straw mushroom into a crude polysaccharide solution of the straw mushroom;

[0024] (8) After soaking and cleaning the 5 kDa dialysis bag, add the crude polysaccharide solution of Volvariella volvacea, seal it, and dialyze it at 4 °C for 24 hours using PBS buffer to collect the polysaccharide solution of Volvariella volvacea;

[0025] (9) Concentrating the Volvariella volvacea polysaccharide solution to 20% of the original volume using a rotary evaporator to obtain a concentrated Volvariella volvacea polysaccharide solution;

[0026] (10) The concentrated Volvariella volvacea polysaccharide solution is vacuum freeze-dried to obtain Volvariella volvacea polysaccharide.

[0027] Preferably, the anti-aging repair is the repair of skin photoaging damage caused by ultraviolet rays.

[0028] Secondly, the present invention provides a method for preparing a stem cell exosome preparation for anti-aging and repair, and the preparation method for each 100g of the stem cell exosome preparation comprises the following steps:

[0029] (1) Add 0.8 g of carbomer to 80 g of distilled water and stir until completely dissolved to form a uniform translucent solution;

[0030] (2) Adjust the pH to 7, add 3 g of glycerol, and stir evenly;

[0031] (3) Add 0.2 g sodium hyaluronate and continue stirring until completely dissolved;

[0032] (4) adding 0.5 g of phenoxyethanol and stirring evenly to obtain a gel matrix;

[0033] (5) Adding the polysaccharide of Volvariella volvacea and the stem cell exosomes into the gel matrix, supplementing with distilled water to make the total weight 100 g, and gently stirring to obtain the stem cell exosome gel preparation.

[0034] Preferably, the stem cell exosomes are adipose-derived mesenchymal stem cell exosomes;

[0035] The Volvariella volvacea polysaccharide is prepared by the preparation method described in claim 2.

[0036] Preferably, the anti-aging repair is the repair of skin photoaging damage caused by ultraviolet rays.

[0037] In addition, the present invention provides an application of a composition in the preparation of an anti-aging and repairing gel preparation, wherein the composition is stem cell exosomes and Volvariella volvacea polysaccharide, and the mass ratio of the stem cell exosomes to the Volvariella volvacea polysaccharide is 1:4.

[0038] Preferably, the stem cell exosomes are adipose-derived mesenchymal stem cell exosomes;

[0039] The Volvariella volvacea polysaccharide is prepared by the preparation method described in claim 2.

[0040] Preferably, the anti-aging repair is the repair of skin photoaging damage caused by ultraviolet rays.

[0041] Preferably, in every 100 g of the gel preparation, the content of the stem cell exosomes is 0.002 g, and the content of the Volvariella volvacea polysaccharide is 0.008 g.

[0042] The beneficial effects of the present invention are:

[0043] 1. The preparation prepared by the present invention exhibits an excellent protective effect in the ultraviolet-induced skin photoaging model, which is specifically manifested in significantly increasing the level of SOD (superoxide dismutase) in skin tissue, reducing the content of MDA (malondialdehyde), alleviating oxidative stress and lipid peroxidation damage, and thus effectively repairing skin damage caused by ultraviolet rays.

[0044] 2. The present invention combines adipose-derived mesenchymal stem cell exosomes with Volvariella polysaccharide in a mass ratio of 1:4 to form an exosome preparation for anti-aging and repair. The results of the study showed that the combination of the two can exert a significant synergistic effect, and can more effectively improve the survival rate of skin cells compared with the use of exosomes or Volvariella polysaccharide alone.

[0045] 3. The present invention adopts a specific alkaline extraction and dialysis purification method to prepare the polysaccharide of Volvariella volvacea, which retains the biological activity of the polysaccharide and removes impurities that may affect the synergistic effect. Compared with the traditional water extraction method, the obtained polysaccharide of Volvariella volvacea exhibits obvious synergistic effect when used in combination with exosomes.

[0046] In summary, the present invention provides an anti-aging and repair preparation with significant synergistic effects by combining adipose-derived mesenchymal stem cell exosomes with Volvariella volvacea polysaccharide, which overcomes the shortcomings of limited effects of a single ingredient, significantly improves the antioxidant, anti-inflammatory and cell repair capabilities, and has good clinical application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The effect of exosomes combined with polysaccharides from Volvariella volvacea on the survival rate of skin cells after UV treatment;

[0048] Figure 2 The effect of exosomes combined with crude polysaccharides of Volvariella volvacea on the survival rate of skin cells treated with ultraviolet light;

[0049] Figure 3 The effects of different gel preparations on the level of SOD (superoxide dismutase) in mouse skin after ultraviolet radiation;

[0050] Figure 4 Effects of different gel preparations on MDA (malondialdehyde) levels in mouse skin after ultraviolet radiation. DETAILED DESCRIPTION

[0051] In order to clearly illustrate the technical features of this solution, this solution is described below through a specific implementation method.

[0052] Example 1

[0053] Preparation of stem cell exosomes

[0054] (1) Collect the supernatant of adipose-derived mesenchymal stem cells cultured for 72 h, centrifuge at 300 g for 10 min, and collect the supernatant;

[0055] (2) Centrifuge at 2000 g for 20 min and collect the supernatant;

[0056] (3) Centrifuge at 10,000 g for 30 min and collect the supernatant;

[0057] (4) Filter the supernatant using a 0.22 μm filter to further remove residual particles;

[0058] (5) Centrifuge at 100,000 g for 120 min, remove the supernatant, and resuspend the precipitate in PBS;

[0059] (6) Centrifuge at 100,000 g for 70 minutes, remove the supernatant, resuspend in PBS, and determine the concentration using the BCA method for later use.

[0060] Example 2

[0061] Preparation of Volvariella polysaccharide

[0062] (1) vacuum freeze-drying the straw mushrooms and then crushing them into fine powder to obtain straw mushroom fine powder A;

[0063] (2) Extract the straw mushroom powder with 80% ethanol at 50°C for three times to remove lipid substances;

[0064] (3) After filtering, vacuum freeze drying is performed to obtain straw mushroom fine powder B;

[0065] (4) adding the straw mushroom powder B into a 0.2 M NaOH solution at a solid-liquid ratio of 1:20, heating to 50° C., and performing alkaline extraction for 3 hours with continuous stirring;

[0066] (5) After adjusting the pH of the extract to neutral, centrifuge at 8000 rpm for 15 min and collect the supernatant;

[0067] (6) Add 4 times the volume of anhydrous ethanol to the supernatant and let it stand for 12 hours;

[0068] (7) collecting the precipitate by centrifugation to obtain crude polysaccharide of Volvariella volvacea, and after drying, preparing the crude polysaccharide of Volvariella volvacea into a crude polysaccharide solution of 1 mg / ml;

[0069] (8) After soaking and cleaning the 5 kDa dialysis bag, add the polysaccharide solution, seal it, and dialyze it at 4 °C for 24 hours using PBS buffer to collect the polysaccharide solution;

[0070] (9) Concentrating the Volvariella volvacea polysaccharide solution to 20% of the original volume using a rotary evaporator to obtain a concentrated Volvariella volvacea polysaccharide solution;

[0071] (10) Vacuum freeze drying to obtain Volvariella volvacea polysaccharide.

[0072] Comparative Example 1

[0073] (1) freeze-drying the straw mushrooms in a vacuum oven and then grinding them into fine powder to obtain straw mushroom fine powder A;

[0074] (2) Extract the straw mushroom powder with 80% ethanol at 50°C for three times to remove lipid substances;

[0075] (3) After filtering, vacuum freeze drying is performed to obtain straw mushroom fine powder B;

[0076] (4) adding the straw mushroom powder B into distilled water at a solid-liquid ratio of 1:20, heating to 50° C., and performing alkaline extraction for 3 hours, with continuous stirring during the process;

[0077] (5) After adjusting the extract to neutral, centrifuge at 8000 rpm for 15 min and collect the supernatant;

[0078] (6) Add 4 times the volume of anhydrous ethanol to the supernatant and let it stand for 12 hours;

[0079] (7) The precipitate is collected by centrifugation and dried to obtain crude polysaccharide of Volvariella volvacea.

[0080] Example 3

[0081] Detection of the protective effect of exosomes combined with polysaccharides from Volvariella volvacea on UV-induced epidermal cell damage

[0082] 1. Cell Culture

[0083] Cell line: immortalized human epidermal keratinocytes (HaCaT cells).

[0084] Culture conditions:

[0085] Culture medium: high glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin.

[0086] Culture environment: cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0087] 2. Cell Passaging and Preparation of Cell Suspension

[0088] Cell digestion:

[0089] (1) When HaCaT cells grow to 80-90% confluence, the culture medium is discarded and the cells are washed twice with a calcium- and magnesium-free PBS solution to remove residual culture medium and metabolites.

[0090] (2) Add an appropriate amount of 0.25% trypsin-EDTA digestion solution and digest at 37°C for 3 minutes. Observe under a microscope. When the cells become round and loosely fall off, immediately add serum-containing culture medium to stop digestion.

[0091] Prepare cell suspension:

[0092] (1) Collect the cell suspension after digestion, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0093] (2) Resuspend the cells in fresh DMEM complete medium and mix by pipetting to make a single-cell suspension.

[0094] (3) Count the cells and adjust the cell density to approximately 1×105 cells / mL.

[0095] 3. Cell Seeding

[0096] (1) The cell suspension was inoculated into a 96-well culture plate at a volume of 100 μL per well, with each well containing approximately 1×104 cells.

[0097] (2) The culture plate was placed in a 37°C, 5% CO2 incubator and incubated until the cells adhered to the wall and grew, and the cell confluence reached more than 85%.

[0098] 4. Experimental Grouping and Treatment

[0099] According to different treatment methods, the cells were divided into the following nine groups, with three replicate wells in each group:

[0100] Con group: The cells were replaced with fresh DMEM complete medium without any special treatment.

[0101] Model group: The cells were replaced with fresh DMEM complete medium and then irradiated with ultraviolet light to simulate cell damage.

[0102] Exosome group: The medium was replaced with DMEM complete medium containing 100 μg / mL exosomes.

[0103] Polysaccharide group: The medium was replaced with DMEM complete medium containing 100 μg / mL Volvariella polysaccharide.

[0104] Combination group a: replaced with DMEM complete medium containing 50 μg / mL exosomes and 50 μg / mL Volvariella polysaccharide.

[0105] Combination group b: replaced with DMEM complete medium containing 40 μg / mL exosomes and 60 μg / mL Volvariella polysaccharide.

[0106] Combination group c: replaced with DMEM complete medium containing 20 μg / mL exosomes and 80 μg / mL Volvariella polysaccharide.

[0107] Combination group d: replaced with DMEM complete medium containing 60 μg / mL exosomes and 40 μg / mL Volvariella polysaccharide.

[0108] Combination group e: replaced with DMEM complete medium containing 80 μg / mL exosomes and 20 μg / mL Volvariella polysaccharide.

[0109] Treatment method:

[0110] (1) Discard the original culture medium of each group of cells and add the corresponding treatment culture medium.

[0111] (2) Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0112] 5. Ultraviolet radiation treatment

[0113] (1) After 24 hours of treatment, discard the culture medium in each well and gently wash the cells once with PBS buffer to remove residual culture medium components.

[0114] (2) Add 100 μL PBS to keep the cells moist and prevent them from drying out.

[0115] (3) Under sterile conditions, place the 96-well plate under an ultraviolet lamp at a distance of 15-20 cm for 5 minutes to evenly irradiate each group of cells to simulate ultraviolet damage.

[0116] 6. Cell Recovery and Continued Culture

[0117] (1) After UV radiation, discard PBS immediately to avoid prolonged nutrient deprivation of cells.

[0118] (2) Fresh DMEM complete medium was added to each group of cells, 100 μL per well.

[0119] (3) Incubate the cells in a 37°C, 5% CO2 incubator for 24 hours to observe the repair and survival of the cells.

[0120] 7. Cell viability assay (CCK-8 method)

[0121] (1) Add CCK-8 reagent: After 24 hours of incubation, add 10 μL of CCK-8 reagent to each well and gently shake to mix to avoid bubbles.

[0122] (2) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 1-2 hours until the control group shows obvious color.

[0123] (3) The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader, and the cell survival rate was calculated. The results are shown in Table 1 and Figure 1 shown.

[0124] Table 1 Effect of exosomes combined with Volvariella volvacea polysaccharide on cell survival rate of skin cells treated with UV

[0125]

[0126]

[0127] From Table 1 and Figure 1 It can be seen that compared with the Con group, the cell survival rate of the Model group decreased significantly, indicating that the present invention successfully simulated the photoaging damage caused by ultraviolet rays;

[0128] Compared with the Model group, the cell survival rates of the other groups supplemented with exosomes and Volvariella polysaccharide were significantly increased, indicating that the adipose-derived mesenchymal stem cell exosomes and Volvariella polysaccharide prepared by the present invention, when used alone, can effectively protect against photoaging damage caused by ultraviolet rays.

[0129] For the combined group, it can be seen that when the total amount of addition remains unchanged, only the cell survival rate of the combined group c is significantly improved compared with the exosome group and the polysaccharide group. The possible reason is that the straw mushroom polysaccharide provides a strong antioxidant and anti-inflammatory protection, and the exosome provides a moderate repair and regeneration ability. When the mass ratio of straw mushroom polysaccharide and exosome is 4:1, the best balance between the two is achieved, which can effectively reduce the damage of ultraviolet rays to cells and promote repair after damage. However, due to the imbalance of the concentration of exosomes or straw mushroom polysaccharides in other combined groups, the antioxidant or repair capacity is insufficient or excessive, and a significant synergistic effect cannot be exerted.

[0130] Example 4

[0131] To test whether the combination of exosomes and crude polysaccharides from Volvariella volvacea has a synergistic effect on UV-induced epidermal cell damage

[0132] 1. Cell Culture

[0133] Cell line: immortalized human epidermal keratinocytes (HaCaT cells).

[0134] Culture conditions:

[0135] Culture medium: high glucose DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin.

[0136] Culture environment: cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0137] 2. Cell Passaging and Preparation of Cell Suspension

[0138] Cell digestion:

[0139] (1) When HaCaT cells grow to 80-90% confluence, the culture medium is discarded and the cells are washed twice with a calcium- and magnesium-free PBS solution to remove residual culture medium and metabolites.

[0140] (2) Add an appropriate amount of 0.25% trypsin-EDTA digestion solution and digest at 37°C for 3 minutes. Observe under a microscope. When the cells become round and loosely fall off, immediately add serum-containing culture medium to stop digestion.

[0141] Prepare cell suspension:

[0142] (1) Collect the cell suspension after digestion, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0143] (2) Resuspend the cells in fresh DMEM complete medium and mix by pipetting to make a single-cell suspension.

[0144] (3) Count the cells and adjust the cell density to approximately 1×105 cells / mL.

[0145] 3. Cell Seeding

[0146] (1) The cell suspension was inoculated into a 96-well culture plate at a volume of 100 μL per well, with each well containing approximately 1×104 cells.

[0147] (2) The culture plate was placed in a 37°C, 5% CO2 incubator and incubated until the cells adhered to the wall and grew, and the cell confluence reached more than 85%.

[0148] 4. Experimental Grouping and Treatment

[0149] According to different treatment methods, the cells were divided into the following five groups, with three replicate wells in each group:

[0150] Con group: The cells were replaced with fresh DMEM complete medium without any special treatment.

[0151] Model group: The cells were replaced with fresh DMEM complete medium and then irradiated with ultraviolet light to simulate cell damage.

[0152] Exosome group: The medium was replaced with DMEM complete medium containing 100 μg / mL exosomes.

[0153] Crude polysaccharide group: The medium was replaced with DMEM complete medium containing 100 μg / mL crude polysaccharide of Volvariella volvacea.

[0154] Combination group: replaced with DMEM complete medium containing 20 μg / mL exosomes and 80 μg / mL crude polysaccharide of Volvariella volvacea.

[0155] Treatment method:

[0156] (1) Discard the original culture medium of each group of cells and add the corresponding treatment culture medium.

[0157] (2) Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0158] 5. Ultraviolet radiation treatment

[0159] (1) After 24 hours of treatment, discard the culture medium in each well and gently wash the cells once with PBS buffer to remove residual culture medium components.

[0160] (2) Add 100 μL PBS to keep the cells moist and prevent them from drying out.

[0161] (3) Under sterile conditions, place the 96-well plate under an ultraviolet lamp at a distance of 15-20 cm for 5 minutes to evenly irradiate each group of cells to simulate ultraviolet damage.

[0162] 6. Cell Recovery and Continued Culture

[0163] (1) After UV radiation, discard PBS immediately to avoid prolonged nutrient deprivation of cells.

[0164] (2) Fresh DMEM complete medium was added to each group of cells, 100 μL per well.

[0165] (3) Incubate the cells in a 37°C, 5% CO2 incubator for 24 hours to observe the repair and survival of the cells.

[0166] 7. Cell viability assay (CCK-8 method)

[0167] (1) Add CCK-8 reagent: After 24 hours of incubation, add 10 μL of CCK-8 reagent to each well and gently shake to mix to avoid bubbles.

[0168] (2) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 1-2 hours until the control group shows obvious color.

[0169] (3) The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader, and the cell survival rate was calculated. The results are shown in Table 2 and Figure 2 shown.

[0170] Table 2 Effect of exosomes combined with crude polysaccharides of Volvariella volvacea on the cell survival rate of skin cells treated with UV

[0171] Experimental Grouping Cell survival rate (%) Con Group 100±3.64 Model Group 48.90±2.47 Exosome group 72.29±2.80 Crude polysaccharide group 66.76±3.21 Combination group 64.27±2.46

[0172] From the results in Table 2, it can be seen that compared with the exosome group and the crude polysaccharide group, the combined group did not show significant synergistic effects at the same mass ratio. This may be due to the differences in polysaccharide structure and composition between the polysaccharide extracted by alkali extraction and the polysaccharide extracted by water extraction, which resulted in the polysaccharide having functions that the crude polysaccharide prepared in Example 2 did not have, and the preparation method in Example 1 filtered out some impurities by dialysis, which could make the polysaccharide better exert its effect, resulting in different effects of the polysaccharides prepared in Example 1 and Example 2.

[0173] Example 5

[0174] Preparation of stem cell exosome gel preparation for anti-aging and repair

[0175] 1. Recipe table (100g)

[0176] The following formula was prepared: 0.008 g of Volvariella polysaccharide, 0.002 g of adipose-derived mesenchymal stem cell exosomes, 0.2 g of hyaluronic acid, 3 g of glycerol, 0.8 g of carbomer, 0.5 g of phenoxyethanol, and 95.49 g of water.

[0177] 2. Preparation of Gel Matrix

[0178] (1) Add 0.8 g of carbomer to 80 g of distilled water and stir until completely dissolved to form a uniform translucent solution;

[0179] (2) Adjust the pH to 7, add 3 g of glycerol, and stir evenly;

[0180] (3) Add 0.2 g sodium hyaluronate and continue stirring until completely dissolved;

[0181] (4) Add 0.5 g of phenoxyethanol and stir evenly to obtain a gel matrix.

[0182] 3. Addition of polysaccharides and exosomes

[0183] (1) Adding volvacea polysaccharide and adipose-derived mesenchymal stem cell exosomes into a gel matrix, supplementing with distilled water to make the total weight 100 g, and gently stirring to obtain a stem cell exosome gel preparation.

[0184] (2) The prepared gel preparation is packaged into sterile packaging bottles for later use.

[0185] At the same time, a polysaccharide group gel preparation containing only Volvariella polysaccharide and an exosome gel preparation containing only exosomes were prepared according to the same method.

[0186] Example 6

[0187] Detection of the protective effects of different gel preparations on photoaging-damaged mouse skin

[0188] 1. Selection and feeding of experimental animals

[0189] (1) Thirty BALB / c mice were selected, with a body weight of 20 ± 2 g and an age of 6 to 8 weeks;

[0190] (2) The mice were acclimatized under the same environmental conditions for 1 week. The temperature, humidity, and light cycle were controlled in the breeding environment, and sufficient food and water were provided to ensure that the experimental mice were in a healthy state.

[0191] 2. Anesthesia and Preparation of Mice

[0192] (1) Before the experiment, mice were anesthetized with 3% sodium pentobarbital injected intraperitoneally. The anesthetic dose was calculated based on the weight of the mice to ensure that the mice were in a deep anesthesia state.

[0193] After anesthesia, use surgical scissors to cut the long hair on the back of the mouse, and use a depilatory knife to further remove the fine hair to make the skin smooth for subsequent treatment.

[0194] 3. Chemical hair removal

[0195] (1) Chemical hair removal was performed by applying cotton wool dipped in 6% sodium sulfide solution to the back of the mouse. The application range was 3 cm × 3 cm area on the back.

[0196] (2) After hair removal, carefully remove the sodium sulfide solution remaining on the skin surface to prevent drug residues from affecting subsequent experiments.

[0197] 4. Experimental Grouping

[0198] (1) Mice were randomly divided into 5 groups, with 6 mice in each group. The treatment of each group was as follows:

[0199] Con group: normal saline was applied and no UV irradiation was performed.

[0200] Model group: applied with saline solution and irradiated with ultraviolet light.

[0201] Exosome gel group: Apply gel containing exosomes.

[0202] Polysaccharide gel group: Apply gel containing Volvariella volvacea polysaccharide.

[0203] Combined gel group: Applying a gel containing exosomes and Volvariella volvacea polysaccharide (the stem cell exosome gel preparation prepared by the present invention)

[0204] Wait 30 minutes after applying the gel to ensure that it is fully absorbed.

[0205] 5.UVB irradiation treatment

[0206] (1) Use UVB lamp for UVB irradiation with energy of 300mJ / cm 2 The irradiated area is the back area after hair removal.

[0207] (2) Each mouse was irradiated once a day for 3 consecutive days.

[0208] 6. Mouse Sacrifice and Skin Tissue Collection

[0209] (1) After UVB irradiation, mice were quickly killed by cervical dislocation.

[0210] (2) Use surgical scissors to cut the depilated and irradiated skin area, ensuring that the size of the skin sample for each mouse is consistent to ensure comparability of subsequent experiments.

[0211] 7. Skin tissue processing

[0212] (1) Place the collected skin tissue in pre-cooled saline and rinse the skin sample to remove impurities such as residual blood.

[0213] (2) Use filter paper to gently wipe the skin sample dry, ensuring that there is no excess liquid on the surface of the sample.

[0214] 8. Skin Tissue Homogenization

[0215] (1) Add 9 times the weight of normal saline according to the sample weight to ensure the same dilution ratio in subsequent experiments.

[0216] (2) Cut the skin sample into small pieces and grind them thoroughly in a cryogenic grinder to ensure that the skin tissue is evenly broken.

[0217] (3) The ground skin homogenate was placed in a low-temperature centrifuge and centrifuged at 2000 rpm for 15 minutes to separate the supernatant and solid precipitate.

[0218] 9. Determination of relative content of SOD (superoxide dismutase)

[0219] The supernatant after centrifugation was collected and used to determine the level of SOD (superoxide dismutase) in mouse skin tissue.

[0220] According to the instructions of the SOD kit, follow the steps and use the content of the Model group as the standard to calculate the relative content difference of SOD in each group of skin samples. The results are as follows Figure 3 shown.

[0221] 10. Determination of relative content of MDA

[0222] According to the same method as step 9, the relative content of MDA in the skin samples of each group was detected using the MDA kit. The results were as follows: Figure 4 shown.

[0223] from Figure 3 and Figure 4 From the results, it can be seen that compared with the exosome gel group and the polysaccharide gel group used alone, under the condition of the same total usage, the combined gel group showed a more significant increase in SOD levels and a decrease in MDA levels. Specifically, the SOD (superoxide dismutase) in the combined gel group was more significantly increased than that in the Model group, indicating that its antioxidant capacity was stronger and effectively reduced the oxidative stress caused by ultraviolet radiation. At the same time, the MDA (malondialdehyde) level in the combined gel group was significantly lower than that in the Model group, indicating that the degree of lipid peroxidation was significantly reduced, further verifying its ability to resist ultraviolet damage. The results show that the gel preparation prepared by the present invention in combination with stem cell exosomes and straw mushroom polysaccharides has a more superior effect in protecting against photoaging caused by ultraviolet rays. This shows that the preparation has potential clinical application value, especially in the fields of anti-aging and skin repair.

Claims

1. A stem cell exosome preparation for anti-aging and repair, characterized in that: Each 100g of the stem cell exosome preparation contains the following components: Stem cell exosomes: 0.002g, Volvariella polysaccharide: 0.008g, Sodium hyaluronate: 0.2g, Glycerin: 3g, Carbomer: 0.8g, Phenoxyethanol: 0.5g, Distilled water: up to 100g.

2. The stem cell exosome preparation according to claim 1, characterized in that The stem cell exosomes are adipose mesenchymal stem cell exosomes; The straw mushroom polysaccharide is prepared by the following preparation method: (1) freeze-drying the straw mushrooms in a vacuum oven and then grinding them into fine powder to obtain straw mushroom fine powder A; (2) refluxing the straw mushroom powder with ethanol to remove lipid substances; (3) After filtering, vacuum freeze drying is performed to obtain straw mushroom fine powder B; (4) adding the straw mushroom powder B into a 0.2 M NaOH solution at a solid-liquid ratio of 1:20, heating to 50° C., and performing alkaline extraction for 3 hours with continuous stirring; (5) After adjusting the pH of the extract to neutral, centrifuge and collect the supernatant; (6) Add 4 times the volume of anhydrous ethanol to the supernatant and let it stand for 12 hours; (7) collecting the precipitate by centrifugation, drying it, and obtaining crude polysaccharide of the straw mushroom, and preparing the crude polysaccharide of the straw mushroom into a crude polysaccharide solution of the straw mushroom; (8) After soaking and cleaning the 5 kDa dialysis bag, add the crude polysaccharide solution of Volvariella volvacea, seal it, and dialyze it at 4 °C for 24 hours using PBS buffer to collect the polysaccharide solution of Volvariella volvacea; (9) Concentrating the Volvariella volvacea polysaccharide solution to 20% of the original volume using a rotary evaporator to obtain a concentrated Volvariella volvacea polysaccharide solution; (10) The concentrated Volvariella volvacea polysaccharide solution is vacuum freeze-dried to obtain Volvariella volvacea polysaccharide.

3. The stem cell exosome preparation according to claim 2, characterized in that: The anti-aging repair is the repair of skin photoaging damage caused by ultraviolet rays.

4. A method for preparing a stem cell exosome preparation for anti-aging and repair, characterized in that: The preparation method of each 100g of the stem cell exosome preparation comprises the following steps: (1) Add 0.8 g of carbomer to 80 g of distilled water and stir until completely dissolved to form a uniform translucent solution; (2) Adjust the pH to 7, add 3 g of glycerol, and stir evenly; (3) Add 0.2 g sodium hyaluronate and continue stirring until completely dissolved; (4) adding 0.5 g of phenoxyethanol and stirring evenly to obtain a gel matrix; (5) Adding the polysaccharide of Volvariella volvacea and the stem cell exosomes into the gel matrix, supplementing with distilled water to make the total weight 100 g, and gently stirring to obtain the stem cell exosome gel preparation.

5. The preparation method according to claim 4, characterized in that: The stem cell exosomes are adipose mesenchymal stem cell exosomes; The Volvariella volvacea polysaccharide is prepared by the preparation method described in claim 2.

6. The preparation method according to claim 5, characterized in that: The anti-aging repair is the repair of skin photoaging damage caused by ultraviolet rays.

7. Use of a composition in the preparation of an anti-aging and repairing gel preparation, characterized in that: The composition is stem cell exosomes and Volvariella volvacea polysaccharide. Calculated by mass ratio, the mass ratio of the stem cell exosomes to the Volvariella volvacea polysaccharide is 1:

4.

8. The use according to claim 7, characterized in that: The stem cell exosomes are adipose mesenchymal stem cell exosomes; The Volvariella volvacea polysaccharide is prepared by the preparation method described in claim 2.

9. The use according to claim 8, characterized in that: The anti-aging repair is the repair of skin photoaging damage caused by ultraviolet rays.

10. The use according to claim 9, characterized in that: In every 100 g of the gel preparation, the content of the stem cell exosomes is 0.002 g, and the content of the Volvariella volvacea polysaccharide is 0.008 g.

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