Application of external vesicles of oral-cavity-derived mesenchymal stem cells in preparation of medicines for repairing skin exogenous aging

Abstract

The invention provides application of oral-cavity-derived mesenchymal stem cell outer vesicles in preparation of a medicine for repairing skin exogenous aging, and relates to the technical field of cell biology. By researching the application effect of the oral-cavity-derived mesenchymal stem cell outer vesicles in repairing aging damage caused by skin exogenous light aging, the obtained oral-cavity-derived mesenchymal stem cell outer vesicles can effectively repair aging and damage caused by skin exogenous light aging. Specifically, a skin light aging model group and stem cell outer vesicles of other sources are obtained by applying a pharmaceutical unit dose of oral mesenchymal stem cell outer vesicles to a skin light aging model obtained by irradiating human skin fibroblasts with a UVb lamp; the external vesicle group of the oral-cavity-derived mesenchymal stem cells has a better skin improving effect, so that a new thought is provided for researching and developing medicines capable of effectively preventing and treating skin aging or injury caused by light aging.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to an application of oral mesenchymal stem cell extracellular vesicles in the preparation of a drug for repairing exogenous skin aging. Background Art

[0002] Exogenous aging is also called photoaging. The main component of sunlight is ultraviolet rays. When exposed to ultraviolet rays, the skin will produce a large amount of reactive oxygen species (ROS), which will lead to lipid peroxidation, enhanced oxidative stress response in skin cells, damage the integrity of cell membranes and the antioxidant system in cells, and further cause cell inflammation and apoptosis. At the same time, the ROS produced by ultraviolet rays will also stimulate cells to produce cytokines, which will activate melanocytes and darken the skin. In addition, ultraviolet rays will increase the synthesis of matrix metalloproteinases (MMPs) in cells, enhance the effects of hyaluronidase and elastase, and intensify the degradation of collagen and elastin, resulting in reduced skin elasticity and wrinkles.

[0003] At present, the main methods to fight skin aging include daily skin care, sun protection, and medical cosmetic treatments. Specifically, although the aged cuticle on the surface of the skin can be removed and the skin can be replenished with moisture and nutrients during daily skin care, it cannot effectively alleviate the exogenous aging caused by photodamage. Although the chemical ingredients in sunscreen products can block ultraviolet rays, they are not easily absorbed by the skin and have no repair function. They cannot repair existing photodamaged skin. As for medical cosmetic treatments, physical stimulation and energy are usually used to improve skin aging, which may cause local skin tissue to be frustrated, resulting in tissue fluid exudation, swelling and discomfort; people with sensitive or weak skin may damage the skin's natural barrier when using medical cosmetic treatments, making the skin sensitive and dry, and accelerating skin aging.

[0004] It can be seen that the existing anti-skin aging methods cannot effectively intervene or prevent the damage caused by exogenous aging to the skin from the inside of the skin cells, and may even lead to skin infections and complications. Therefore, it is necessary and urgent to research and develop a new method that can effectively prevent and treat skin aging or damage caused by photoaging.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to confirm the application effect of oral mesenchymal stem cell extracellular vesicles in the treatment of exogenous skin aging or damage caused by photoaging through research, and on this basis, explore the research and development and clinical transformation of oral mesenchymal stem cell extracellular vesicle drugs, so as to facilitate the development of highly effective drugs for alleviating skin photoaging, which has important scientific research significance and clinical application prospects.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The present invention provides an application of oral mesenchymal stem cell extracellular vesicles in the preparation of a drug for repairing exogenous skin aging.

[0009] Furthermore, the oral-derived mesenchymal stem cell extracellular vesicles are secreted by mesenchymal stem cells from the dental pulp of shed deciduous teeth;

[0010] Alternatively, the oral mesenchymal stem cell extracellular vesicles are secreted by gingival mesenchymal stem cells.

[0011] Furthermore, the positive rates of the cell surface markers CD105, CD73, CD90 and CD29 of the oral-derived mesenchymal stem cells are all ≥95%, and the positive rates of CD11b, CD34, CD45 and HLA-DR are all ≤2%.

[0012] Furthermore, the extrinsic skin aging is skin aging or damage caused by photoaging.

[0013] Furthermore, the application is to administer a unit dose of oral-derived mesenchymal stem cell extracellular vesicles.

[0014] Furthermore, the administration method of the application is to inject a unit dose of oral mesenchymal stem cell extracellular vesicles subcutaneously.

[0015] Furthermore, the unit dose of the subcutaneous injection is 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles in each μg is 1×10 8 ~1.5x10 8 .

[0016] The present invention provides a medicine for repairing exogenous skin aging, which comprises oral mesenchymal stem cell extracellular vesicles and pharmaceutically acceptable excipients.

[0017] Furthermore, the unit dosage of the oral mesenchymal stem cell extracellular vesicles in the drug is 20 μg / mL.

[0018] Furthermore, the dosage form of the exogenous skin aging repair drug includes at least one of an injection, an injection, and a lyophilized agent.

[0019] Furthermore, the drug is an injection, the unit dose of the injection is 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles in each μg is 1x10 8 ~1.5x10 8 .

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides an application of extracellular vesicles of oral mesenchymal stem cells in the preparation of a drug for repairing exogenous skin aging. This application confirms the application effect of extracellular vesicles of oral mesenchymal stem cells in the treatment of exogenous skin aging or damage caused by photoaging through research, and on this basis explores the research and development and clinical transformation of extracellular vesicle drugs of oral mesenchymal stem cells.

[0022] The present invention provides a drug for repairing exogenous skin aging, which comprises oral mesenchymal stem cell extracellular vesicles and pharmaceutically acceptable excipients. According to experiments, oral mesenchymal stem cell extracellular vesicles at a pharmaceutical unit dose can effectively repair exogenous skin aging or damage caused by photoaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a cell morphology diagram of deciduous tooth pulp mesenchymal stem cells provided in Example 1 of the present invention;

[0025] Figure 2 This is a graph showing the cell surface marker detection results provided in Example 1 of the present invention;

[0026] Figure 3 This is an electron transmission microscope image of the extracellular vesicles of oral mesenchymal stem cells provided in Example 2 of the present invention;

[0027] Figure 4 This is a diagram showing the NTA results of extracellular vesicles of oral mesenchymal stem cells provided in Example 2 of the present invention;

[0028] Figure 5 A comparison chart of CCK-8 test results of the control group, injury group and drug administration group provided in Example 3 of the present invention;

[0029] Figure 6 A comparison chart of the EdU-cell cycle detection results of the injury group and the drug-administered group provided in Example 3 of the present invention;

[0030] Figure 7 This is a SA-β-Gal staining result diagram of the control group, injury group and drug administration group provided in Example 3 of the present invention;

[0031] Figure 8A bar chart comparing the SA-β-Gal staining results of the control group, injury group and drug-treated group provided in Example 3 of the present invention;

[0032] Figure 9a The Annexin V-PI apoptosis test results of each experimental group provided in Example 3 of the present invention;

[0033] Figure 9b A bar chart comparing the results of the Annexin V-PI apoptosis experiment of each experimental group provided in Example 3;

[0034] Fig.10 A comparison chart of the ROS reactive oxygen species detection results of each experimental group provided in Example 3;

[0035] Fig.11 A quantitative statistical graph of the ROS reactive oxygen species detection results of each experimental group provided in Example 3;

[0036] Fig.12a The JC-1 mitochondrial membrane potential detection results of each experimental group provided in Example 3;

[0037] Figure 12b A bar chart comparing the JC-1 mitochondrial membrane potential detection results of each experimental group provided in Example 3;

[0038] Fig.13a The mRNA expression level results of the aging-related genes P15 and P16 provided in Example 3 of the present invention;

[0039] Fig.13b The mRNA expression level results of the aging-related genes P19 and P21 provided in Example 3 of the present invention;

[0040] Fig.13c The mRNA expression level results of the aging-related genes P27 and MMP1 provided in Example 3 of the present invention;

[0041] Fig.13d The mRNA expression level results of the aging-related genes MMP2 and MMP3 provided in Example 3 of the present invention;

[0042] Fig.14 A graph showing the expression levels of aging-related proteins in each experimental group provided in Example 3 of the present invention;

[0043] Fig.15 This is a result graph of the mRNA expression level of aging-related genes provided in Example 4 of the present invention;

[0044] Fig.16 A graph showing the JC-1 mitochondrial membrane potential detection results of each experimental group provided in Example 4 of the present invention;

[0045] Fig.17 This is a picture showing the effect of improving the facial condition of a patient after subcutaneous facial injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells, provided in Example 5 of the present invention;

[0046] Fig.18 Another picture showing the improvement of the patient's facial condition after subcutaneous facial injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells provided in Example 5 of the present invention;

[0047] Fig.19 This is a picture showing the effect of improving the condition of the tail of the eye after subcutaneous injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells into the face, provided in Example 5 of the present invention;

[0048] Fig. 20 This is a picture provided in Example 5 of the present invention showing the effect of using extracellular vesicles of deciduous tooth pulp mesenchymal stem cells for subcutaneous facial injection to reduce acne marks and promote the recovery of acne skin. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0050] According to one aspect of the present invention, an oral mesenchymal stem cell extracellular vesicle is used in the preparation of a drug for repairing exogenous skin aging.

[0051] The present invention provides an application of oral mesenchymal stem cell extracellular vesicles in the preparation of a drug for repairing exogenous skin aging. This application confirms the application effect of oral mesenchymal stem cell extracellular vesicles in the treatment of exogenous skin aging or damage caused by photoaging through research, and on this basis explores the research and development and clinical transformation of oral mesenchymal stem cell extracellular vesicle drugs, so as to facilitate the development of highly effective drugs for alleviating skin photoaging.

[0052] It should be noted that mesenchymal stem cells are gradually used for cell transplantation therapy of various diseases due to their strong directional differentiation ability, self-renewal ability and paracrine function. Oral mesenchymal stem cells have been a hot topic in regenerative medicine in recent years. They have shown great potential in tissue regeneration, repair and disease treatment. Compared with embryonic stem cells and induced pluripotent stem cells, they are less controversial ethically, and their derived extracellular vesicles are easier to obtain. Extracellular vesicles (EVs) are lipid bilayer nanoparticles released by cells, rich in a variety of bioactive substances, including proteins, nucleic acids, peptides, etc. These components carry out intercellular communication, signal transduction, and gene expression regulation between skin-related cells through extracellular vesicles, thereby improving the cell viability of skin cells from the root. Compared with stem cells, extracellular vesicles have low immunogenicity and can avoid immune rejection reactions in the body; under appropriate storage conditions, they can maintain their activity and stability for a long time and are not easy to lose bioactive substances. However, there have been no relevant research reports on the study of oral mesenchymal stem cell extracellular vesicles in the preparation of exogenous aging drugs for repairing skin.

[0053] In a preferred embodiment of the present invention, the oral cavity-derived mesenchymal stem cell extracellular vesicles are secreted by mesenchymal stem cells from the pulp of exfoliated deciduous teeth; or, the oral cavity-derived mesenchymal stem cell extracellular vesicles are secreted by mesenchymal stem cells from gingival mesenchymal stem cells.

[0054] As a preferred embodiment, the present application has obtained through experiments that oral-derived mesenchymal stem cell extracellular vesicles are not only easier to obtain than embryonic stem cells, induced pluripotent stem cells, and umbilical cord and adipose-derived mesenchymal stem cell extracellular vesicles, but also have better photodamage repair effects in the human skin fibroblast photoaging model verification test.

[0055] In a preferred embodiment of the present invention, the positive rates of the cell surface markers CD105, CD73, CD90 and CD29 of the oral-derived mesenchymal stem cells are all ≥95%, and the positive rates of CD11b, CD34, CD45 and HLA-DR are all ≤2%.

[0056] In a preferred embodiment of the present invention, the exogenous skin aging is skin aging or damage caused by photoaging.

[0057] In a preferred embodiment of the present invention, the application is to administer a unit dose of oral cavity-derived mesenchymal stem cell extracellular vesicles, and the administration method of the application is to administer a unit dose of oral cavity-derived mesenchymal stem cell extracellular vesicles by subcutaneous injection.

[0058] In the above preferred embodiment, the unit dose for subcutaneous injection is 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles in each μg is 1×10 8~1.5x10 8 .

[0059] According to one aspect of the present invention, a drug for repairing exogenous skin aging comprises oral mesenchymal stem cell extracellular vesicles and pharmaceutically acceptable excipients.

[0060] The present invention provides a drug for repairing exogenous skin aging, which comprises oral mesenchymal stem cell extracellular vesicles and pharmaceutically acceptable excipients. According to experiments, oral mesenchymal stem cell extracellular vesicles at a pharmaceutical unit dose can effectively repair exogenous skin aging or damage caused by photoaging.

[0061] In a preferred embodiment of the present invention, the unit dosage of the oral mesenchymal stem cell extracellular vesicles in the medicine is 20 μg / mL.

[0062] In a preferred embodiment of the present invention, the dosage form of the exogenous skin aging repair drug includes at least one of an injection, an injection, and a lyophilized agent.

[0063] In the above preferred embodiment, the drug is an injection, the unit dose of the injection is 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles per μg is 1×10 8 ~1.5x10 8 .

[0064] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0065] Example 1 Preparation of oral mesenchymal stem cells

[0066] In this embodiment, oral mesenchymal stem cells are prepared by taking deciduous tooth pulp mesenchymal stem cells as an example.

[0067] (I) The specific preparation method is as follows:

[0068] 1. Primary culture of deciduous tooth pulp mesenchymal stem cells: Take the deciduous teeth that have fallen out from healthy people, remove their pulp after cleaning, cut the pulp into pieces and digest it, centrifuge it, discard the supernatant, resuspend it in primary mesenchymal culture medium and then inoculate and culture it. After 4 days, discard the supernatant, add new culture medium and continue to culture. When the cells form larger clones, they can be passaged.

[0069] 2. Subculture of deciduous tooth pulp mesenchymal stem cells: Take the primary cells obtained above, discard the culture medium, add PBS buffer to rinse the cell growth surface, discard the PBS buffer, digest with trypsin, centrifuge, discard the supernatant, resuspend the cells with mesenchymal culture medium, subculture the cells, and perform indicator detection at the 8th generation. If the detection is qualified, the subsequent operation can be carried out.

[0070] Wherein: the primary mesenchymal culture medium is: 20% FBS by volume + 80% α-MEM by volume. The mesenchymal culture medium is: 10% FBS by volume + 90% α-MEM by volume.

[0071] (ii) The index detections are: cell morphology detection, cell survival rate detection, microbial detection, and flow cytometric identification of mesenchymal stem cell surface markers.

[0072] Qualified cell morphology standard: When cultured on the wall, the fibroblasts are spindle-shaped and fusiform, with uniform morphology.

[0073] Cell survival rate qualification standard: uncryopreserved cell survival rate ≥ 90%, and resuscitated cell survival rate ≥ 90%.

[0074] Microbiological testing pass standard: fungi, bacteria and endotoxins detected by membrane filtration and horseshoe crab reagent are all negative.

[0075] Qualified criteria for cell surface marker detection: CD105, CD73, CD90, CD29 positivity rates ≥ 95%, CD11b, CD34, CD45, HLA-DR positivity rates ≤ 2%.

[0076] Figure 1 Figure 2 is a cell morphology diagram of deciduous tooth pulp mesenchymal stem cells in this example. Figure 1 It can be seen that when the deciduous tooth pulp mesenchymal stem cells are cultured on the wall, they appear as spindle-shaped and fusiform fibroblasts with uniform morphology, which conforms to the standard morphology of mesenchymal stem cells.

[0077] Figure 2 : is the result of the cell surface marker detection in this embodiment. Figure 2 It can be seen that the deciduous tooth pulp mesenchymal stem cells provided in this embodiment highly express the mesenchymal stem cell surface markers CD73 and CD90, and the positive rate is above 99%, which meets the standard characterization of mesenchymal stem cells.

[0078] Example 2 Preparation of extracellular vesicles of oral mesenchymal stem cells

[0079] In this example, the deciduous tooth pulp mesenchymal stem cells prepared in the above example 1 are used as an example to prepare oral mesenchymal stem cell extracellular vesicles.

[0080] (I) The specific preparation method is as follows:

[0081] At the 7th generation, dental pulp stem cells were cultured at 2×10 4 / cm 2The cells were inoculated into a cell culture dish at a density of 70% and cultured. When the cells grew to a density of 70%, the culture medium was discarded, PBS buffer was added to rinse the cell growth surface, the PBS buffer was discarded, serum-free culture medium was replaced, and culture was continued for 48 hours, and the supernatant was collected.

[0082] Take the supernatant of the above serum-free culture medium and centrifuge at 300g for 10 minutes, take the supernatant, centrifuge at 3000g for 10 minutes, take the supernatant, centrifuge at 20000g for 30 minutes, take the supernatant, filter at 0.22μm, take the precipitate and concentrate, add 30% sucrose heavy water to dissolve, centrifuge at 110000g for 70 minutes, take the precipitate, wash the precipitate several times with PBS, centrifuge at 4,000g for 10 minutes after each wash, and obtain oral mesenchymal stem cell extracellular vesicles, store at -80℃ for use, and use for injection if qualified.

[0083] The indexes detected are: extracellular vesicle morphology, quantity, particle size, etc., and the detection methods include: electron transmission microscopy, NTA, etc.

[0084] Figure 3 This is an electron transmission microscope image of the extracellular vesicles of oral mesenchymal stem cells in this example. Figure 3 It can be seen that the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells extracted in this example present a characteristic disc-like shape, which is a typical extracellular vesicle shape.

[0085] Figure 4 This is the NTA result of the extracellular vesicles of oral mesenchymal stem cells in this example. Figure 4 It can be seen that the particle size distribution of the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells extracted in this example is mainly around 100-200nm.

[0086] Example 3: Verification experiment of the anti-aging and antioxidant effects of extracellular vesicles of oral mesenchymal stem cells in a human skin fibroblast photoaging model

[0087] The experimental model of this example was carried out by irradiating HSF cells (human skin fibroblasts) with UVb lamp. The experimental group was divided into three groups: control group, injury group, and drug administration group, among which:

[0088] Damage group (UVb): HSF cells were treated with 2×10 4 / cm 2 After the cells are inoculated into a cell culture dish / six-well plate at a density of 80%, they are irradiated with a UVb lamp at a dose of 100 mJ.

[0089] The specific operation is as follows: in the biosafety cabinet, the cell culture vessel is open, and a UVb ultraviolet lamp is vertically fixed at 10 cm from the cell surface for irradiation for 52 seconds. The UVb ultraviolet lamp model used is: CGKJ-UV090301 (PHILIPS).

[0090] The drug administration group (SHED-EVs) refers to the experimental group in which 20 μg / mL of oral mesenchymal stem cell extracellular vesicles prepared in Example 2 were added to the injury group after the injury model was established by irradiating HSF cells with UVb lamp.

[0091] The drug group was administered in the form of an injection, the unit dose of the injection was 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles in each μg was 1x10 8 ~1.5x10 8 .

[0092] Control group (Normal): HSF cells were treated with 2×10 4 / cm 2 The cells were seeded into cell culture dishes / six-well plates at a high density and cultured synchronously with the other two groups, while a blank control group was provided without injury or additional drug administration.

[0093] Furthermore, this example detects the effect of the extracellular vesicles of oral mesenchymal stem cells through experiments such as CCK-8, EdU and cell cycle, SA-β-Gal staining, Annexin V-PI, ROS reactive oxygen species detection, JC-1 mitochondrial membrane potential detection, oxidative stress and aging-related gene qPCR detection, and WB.

[0094] Note: In the attached figures of each test in this example, UVb is the injury group, SHED-EVs is the drug-administered group, and Normal is the control group.

[0095] (I) CCK-8 detection

[0096] The experimental models of the control group, injury group and drug administration group were incubated in an incubator for 36-48 hours. When the cells grew to an appropriate density, 10 μL of CCK-8 solution was added to each well, and the culture plate was returned to the incubator for further culturing for 2-4 hours. The optical density (OD) value of each well was measured at a wavelength of 450 nm using an ELISA reader. Data analysis was performed, and the results were as follows: Figure 5 shown.

[0097] Figure 5 This is a comparison chart of the CCK-8 experimental results of the control group, injury group and drug administration group in this example.

[0098] Depend on Figure 5It can be seen that in the photoaging model, the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells in the drug-treated group have the effect of promoting the proliferation of damaged cells after photoaging.

[0099] (ii) EdU-cell cycle detection

[0100] In the experimental models of the above injury group and drug administration group, EdU was added and incubated at 37°C for 2 hours, the cells were collected and fixed with 4% formaldehyde for 10-15 minutes, and the cells were washed with PBS. The reaction system was incubated at room temperature in the dark for 30 min, the cells were washed with PBS, and FxCycle TM Violet DNA staining solution was used for staining at room temperature in the dark for 30 minutes. Cells were screened using a 300-mesh nylon mesh and analyzed using a cell flow cytometer.

[0101] Data were collected using flow cytometry and the data analysis results were shown in Figure 6 shown.

[0102] Figure 6 This is a comparison chart of the EdU-cell cycle detection results of the injury group and the drug-treated group in this example.

[0103] Depend on Figure 6 It can be seen that in the photoaging model, the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells have the effect of reversing the aging trend and restoring cell vitality.

[0104] (III) SA-β-Gal staining experiment:

[0105] When the cells grow to an appropriate density, they are divided into groups for light aging / drug treatment, the culture medium is removed, the cells are washed once with PBS, and the fixative is added and fixed at room temperature for 15 minutes. The fixative is removed and the cells are washed three times with PBS, each for 3 minutes. SA-β-Gal staining solution is added to the culture dish and incubated overnight at 37°C.

[0106] Figure 7 The SA-β-Gal staining results of the control group, injury group and drug administration group in this example are shown. Figure 7 In the data, con was the control group, UVb was the injury group, SHED-EVs referred to the drug-treated group (using deciduous tooth pulp mesenchymal stem cells), and GMSC-EVs referred to the drug-treated group using gingival mesenchymal stem cell extracellular vesicles.

[0107] The administration method and unit dose of the SHED-EVs administration group and the GMSC-EVs administration group were the same.

[0108] Figure 8 This is a bar chart comparing the SA-β-Gal staining results of the control group, injury group and drug administration group in this example.

[0109] Depend on Figure 7 , Figure 8 It can be seen that the cells were observed and photographed using an optical microscope. Senescent cells will show obvious blue reaction products after SA-β-Gal staining, while normal cells will not be stained or will be stained lightly. The results of data analysis using Image J are as follows, which shows that the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells and gingival mesenchymal stem cells have anti-aging effects in the photoaging model.

[0110] (IV) Annexin V-PI apoptosis assay

[0111] When the cells grew to an appropriate density, they were divided into groups for injury / drug treatment, and then digested with trypsin and collected. The collected cells were washed twice with PBS buffer and the cell concentration was adjusted to 1x10 6 / mL, add Annexin V antibody and PI dye and incubate in the dark for 15 minutes, wash the stained cells twice with PBS buffer, resuspend in PBS and put them into the machine.

[0112] Figure 9a Graph showing the results of the Annexin V-PI apoptosis experiment of each experimental group in this example.

[0113] Figure 9b This is a bar chart comparing the results of the Annexin V-PI apoptosis experiment of each experimental group in this example.

[0114] The stained cells were analyzed using flow cytometry to detect cell apoptosis. The data analysis results are as follows, indicating that extracellular vesicles of deciduous dental pulp mesenchymal stem cells have an anti-apoptotic effect in the aging model.

[0115] (V) ROS reactive oxygen species detection

[0116] When the cells grow to an appropriate density, they are divided into groups for injury / drug treatment, and then the experiment is carried out according to the instructions of the Bio-Tech Reactive Oxygen Detection Kit (S0033S). The cell culture medium is removed and 1 mL of diluted DCFH-DA is added to each well. Incubate in a 37°C cell culture incubator for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. Trypsin digestion, culture medium termination, centrifugation and discarding of supernatant, resuspend in PBS and then put into the machine.

[0117] Flow cytometry was used to collect data, and the data analysis results showed that the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells and gingival mesenchymal stem cells had antioxidant effects in the photoaging model.

[0118] Fig.10 A comparison chart of the ROS reactive oxygen species detection results of each experimental group provided in this example.

[0119] Fig.11 This is a quantitative statistical chart of the ROS reactive oxygen species detection results of each experimental group provided in this example.

[0120] (VI) JC-1 mitochondrial membrane potential detection

[0121] When the cells grow to an appropriate density, they are divided into groups for injury / drug treatment, and then the experiment is carried out according to the instructions of the Biological Sciences JC-1 Detection Kit (#551302). The cell culture medium is removed, the cells are washed with PBS, digested and collected, and the JC-1 staining working solution is added and incubated in a 37°C cell culture incubator for 10-15 minutes. The cells are washed three times with buffer and then tested on the machine.

[0122] Flow cytometry was used to collect data, and the data analysis results are as follows, which show that the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells and gingival mesenchymal stem cells have antioxidant effects.

[0123] Fig.12a 1 is the JC-1 mitochondrial membrane potential detection result of each experimental group in this example.

[0124] Figure 12b This is a bar chart comparing the JC-1 mitochondrial membrane potential detection results of each experimental group in this example.

[0125] (VII) Detection of expression levels of aging-related genes

[0126] When the cells grow to an appropriate density, they are divided into groups for light aging / drug treatment, and RNA is extracted using Trizol, chloroform, isopropanol and other solutions. Reverse transcription is then performed to obtain cDNA: using the extracted RNA as a template, reverse transcription is performed using a reverse transcription kit and appropriate primers (such as Oligo (dT) or random primers) to synthesize cDNA. Fluorescence quantitative PCR amplification is then performed using the SYBR Green 20μL reaction system.

[0127] The aging-related gene primers are as follows:

[0128]

[0129]

[0130] Note: In this application, the specific gene names are replaced by their protein abbreviations, as follows: P15 (CDKN2B), P16 (CDKN2A), P19 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), MMP1 (MMP1), MMP2 (MMP2), MMP3 (MMP3).

[0131] The Ct value of each sample was normalized using GAPDH as the reference genome, and each sample was compared with the control group using the established efficiency-based ΔΔCt method. The normalized Ct value was converted to a normalized expression value, and the relative expression of each gene 2^-(ΔΔCt) was calculated, and the graph was analyzed using GraphPad. The data analysis results showed that the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells and gingival mesenchymal stem cells had anti-aging effects.

[0132] Figure 13a to Figure 13d The mRNA expression level results of aging-related genes.

[0133] Fig.13a These are the mRNA expression level results of aging-related genes P15 and P16.

[0134] Fig.13b These are the mRNA expression levels of aging-related genes P19 and P21.

[0135] Fig.13c These are the mRNA expression levels of aging-related genes P27 and MMP1.

[0136] Fig.13d These are the mRNA expression levels of aging-related genes MMP2 and MMP3.

[0137] (VIII) Detection of protein expression levels of aging-related genes

[0138] When the cells grow to a suitable density, they are divided into groups for light aging / drug addition, etc., and then the cells are treated and collected with protein lysis buffer, and then the protein is quantified using the BCA method. After grouping, sample preparation is performed: SDS-PAGE loading buffer is added to the protein sample and heated to 100°C to denature the protein. According to the molecular weight of the target protein, the appropriate separation gel concentration is selected and the concentrated gel is prepared. The protein sample and protein marker are added to the sample loading well of the gel. Electrophoresis is performed under constant pressure or constant current conditions to separate the proteins in the gel according to their molecular weight. Transfer the membrane under constant pressure or constant current conditions to transfer the proteins in the gel to the membrane. The membrane is placed in an incubation box containing a blocking solution and blocked on a shaker at room temperature for 1-2 hours. Then, the primary antibody incubation, membrane washing, secondary antibody incubation, and membrane washing operations are performed, and the color developing solution is added and then exposed.

[0139] The aging-related protein antibody is p16 INK4A (E6N8P) Rabbit mAb (Cell signaling, #18769), and the target protein is qualitatively and semi-quantitatively analyzed according to the position and depth of the band. The data analysis results are as follows, which show that the extracellular vesicles of deciduous tooth pulp mesenchymal stem cells and gingival mesenchymal stem cells have anti-aging effects in the photoaging model.

[0140] Fig.14 Result diagram of aging-related protein expression levels in each experimental group in this example.

[0141] Example 4: Verification experiment on the anti-aging and antioxidant effects of extracellular vesicles of oral mesenchymal stem cells and common umbilical cord and adipose-derived mesenchymal stem cells in the human skin fibroblast photoaging model.

[0142] (I) Extracellular vesicles of oral mesenchymal stem cells were compared with extracellular vesicles of umbilical cord and adipose-derived mesenchymal stem cells to detect the expression levels of genes related to aging and oxidative stress.

[0143] When the cells grow to an appropriate density, they are divided into groups for photoaging / addition of different exosomes, etc., and the expression of aging and oxidative stress related genes is verified using the same RNA extraction, reverse transcription, and qPCR methods as in Example 3. The aging related gene primers are as follows:

[0144]

[0145]

[0146] Fig.15 This is a graph showing the mRNA expression levels of aging-related genes provided in this example.

[0147] Depend on Fig.15 It can be seen that in the photoaging model, at the same dose, extracellular vesicles from oral mesenchymal stem cells have superior anti-aging and antioxidant effects.

[0148] (II) Extracellular vesicles of oral mesenchymal stem cells were compared with extracellular vesicles of umbilical cord and adipose-derived mesenchymal stem cells to detect JC-1 mitochondrial membrane potential

[0149] When the cells grow to an appropriate density, they are divided into groups for injury / drug treatment, and then the experiment is carried out according to the instructions of the Biological Digest JC-1 detection kit (#551302). The cell culture medium is removed, the cells are washed with PBS, digested and collected, and the JC-1 staining working solution is added and incubated in a 37°C cell culture incubator for 10-15 minutes. The cells are washed three times with buffer and then tested on the machine. Data collection is performed using a flow cytometer. The data analysis results are shown in Fig.16 .

[0150] Fig.16 Graph showing the JC-1 mitochondrial membrane potential detection results for each experimental group in this example.

[0151] From the above, it can be seen that in the photoaging model, at the same dose, extracellular vesicles of mesenchymal stem cells derived from the oral cavity have superior anti-aging and antioxidant effects.

[0152] It can be seen from this embodiment that by comparing the extracellular matrix of deciduous tooth pulp mesenchymal stem cells and gingival mesenchymal stem cells in parallel with the common umbilical cord and adipose-derived mesenchymal stem cells, through experiments such as oxidative stress-related gene detection and JC-1 mitochondrial membrane potential detection, it can be observed that oral-derived mesenchymal stem cells have an antioxidant improvement effect on the aging model, and the antioxidant effect is more obvious than that of umbilical cord and adipose-derived mesenchymal stem cells.

[0153] Example 5 Application of facial subcutaneous injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells

[0154] Clean the patient's face and apply anesthetic cream to the injection site. Wash the facial anesthetic cream with clean water after applying the anesthetic cream for about 30-40 minutes. According to the injection dose and site specified in the established plan, a professional physician will perform subcutaneous injection using a water-light machine or hand needle in accordance with the principle of sterility and the water-light needle introduction method. After the injection, apply a postoperative repair mask to promote rapid repair of the needle hole. Specific application effects are as follows: Fig.17 .

[0155] Fig.17 This example provides a picture showing the improvement of a patient's facial condition after subcutaneous facial injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells.

[0156] Fig.18 This is another picture provided in this embodiment showing the improvement of the patient's facial condition after subcutaneous facial injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells.

[0157] Fig.19 This example provides a picture showing the improvement of the tail of the eye after subcutaneous injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells into the face.

[0158] Fig. 20 This example provides a picture of the effect of using extracellular vesicles of deciduous tooth pulp mesenchymal stem cells for subcutaneous facial injection to reduce acne marks and promote the recovery of acne skin.

[0159] Depend on Fig.17 It can be seen that after subcutaneous facial injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells, the patient's facial condition was significantly improved, pores shrank, skin texture became delicate, glossiness increased, and forehead wrinkles and eye wrinkles became lighter.

[0160] Depend on Fig.18 It can be seen that after subcutaneous facial injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells, the patient's facial condition was significantly improved, pigmentation faded, skin color became whiter, pores shrank, skin became delicate, bags under the eyes reduced, wrinkles became lighter, and contours were tighter than before.

[0161] Depend on Fig.19 It can be seen that after subcutaneous injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells around the eyes, the condition of the tail of the eye was significantly improved and wrinkles became lighter.

[0162] Depend on Fig. 20 It can be seen that after subcutaneous injection of extracellular vesicles of deciduous tooth pulp mesenchymal stem cells into the face, the facial condition is significantly improved. It can not only reduce skin redness and inflammation, fade acne marks and promote acne skin recovery, but also improve skin color, make the skin whiter, and lighten spots.

[0163] In summary, the extracellular vesicles derived from deciduous tooth pulp mesenchymal stem cells of the present invention can effectively promote the repair of aging skin, not only improve skin quality, shrink pores, and reduce wrinkles, but also have anti-inflammatory repair effects, improve skin tone, and help repair acne skin.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of extracellular vesicles of oral mesenchymal stem cells in the preparation of drugs for repairing exogenous skin aging.

2. The use according to claim 1, characterized in that: The oral mesenchymal stem cell extracellular vesicles are secreted by mesenchymal stem cells from the dental pulp of shed deciduous teeth; Alternatively, the oral mesenchymal stem cell extracellular vesicles are secreted by gingival mesenchymal stem cells.

3. The use according to claim 2, characterized in that: The positive rates of the cell surface markers CD105, CD73, CD90 and CD29 of the oral-derived mesenchymal stem cells are all ≥95%, and the positive rates of CD11b, CD34, CD45 and HLA-DR are all ≤2%.

4. The use according to claim 1, characterized in that: The exogenous skin aging is skin aging or damage caused by photoaging.

5. The use according to claim 1, characterized in that: The application is to administer a unit dose of oral-derived mesenchymal stem cell extracellular vesicles.

6. The use according to claim 5, characterized in that: The administration method of the application is to inject a unit dose of oral-derived mesenchymal stem cell extracellular vesicles subcutaneously.

7. The use according to claim 6, characterized in that: The unit dose for subcutaneous injection is 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles in each μg is 1x10 8 ~1.5x10 8 .

8. A drug for repairing exogenous skin aging, characterized in that: The medicine comprises oral-derived mesenchymal stem cell extracellular vesicles and pharmaceutically acceptable excipients.

9. The drug for repairing exogenous skin aging according to claim 8, characterized in that: The dosage form of the exogenous skin aging repair drug includes at least one of an injection, an injection, and a lyophilized agent.

10. The drug for repairing exogenous skin aging according to claim 9, characterized in that: The medicine is an injection; The unit dose of the injection is 20 μg / mL, and the content of oral mesenchymal stem cell extracellular vesicles in each μg is 1x10 8 ~1.5x10 8 .

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