Stem cell culture medium and preparation method and application of stem cell exosome with oxidation resistance

Abstract

The invention provides a stem cell culture medium and a preparation method and application of a stem cell exosome with oxidation resistance. The stem cell culture medium comprises baicalin, quercetin, liquiritin and catechin. According to the stem cell culture medium provided by the invention, a mixture of baicalin, quercetin, liquiritin and catechin is adopted as a component of the culture medium to stimulate the stem cells, and the oxidative stress state of the stem cells is changed, so that the stem cells can secrete exosomes with an antioxidant effect, and the secreted exosomes have higher antioxidant capacity. The corresponding stem cell exosome is relatively excellent in the aspect of oxidation resistance.

Description

Technical Field

[0001] The present invention relates to the field of cell-related technologies, and in particular to a stem cell culture medium, a preparation method and application of stem cell exosomes with antioxidant properties. Background Art

[0002] At present, the methods used to combat oxidative damage mainly include the use of antioxidants and lifestyle intervention. Antioxidants can be divided into endogenous antioxidants (such as superoxide dismutase, glutathione peroxidase, etc.) and exogenous antioxidants (such as vitamin C, vitamin E, carotenoids, etc.). Although these antioxidants can remove free radicals and reduce oxidative damage to a certain extent, they also have some limitations.

[0003] Stem cells have always attracted much attention in the field of regenerative medicine. They have the ability of self-renewal and multidirectional differentiation, which provides great potential for the treatment of various diseases. One of the important mechanisms by which stem cells exert their effects is through the secretion of exosomes. Stem cell exosomes are nanoscale vesicles with a diameter of about 30-150nm, which are secreted by stem cells into the extracellular environment. Exosomes contain a variety of bioactive components, such as proteins, nucleic acids (including mRNA, miRNA, etc.) and lipids. These components give exosomes a variety of biological functions, such as regulating intercellular communication, participating in immune regulation, and promoting tissue repair.

[0004] More and more studies have shown that stem cell exosomes can migrate to damaged tissue sites and regulate the functions of target cells by delivering the bioactive substances they contain, bringing new ideas to the treatment of diseases and anti-aging of the body.

[0005] However, the exosomes obtained under traditional culture methods lack targeting and their antioxidant effects are not significant, which seriously affects the clinical application and industrial development of exosomes. Summary of the invention

[0006] The present invention provides a stem cell culture medium, a preparation method and application of stem cell exosomes with antioxidant properties, in order to solve the deficiencies in the prior art.

[0007] As a first aspect of the present invention, the present invention provides a stem cell culture medium with antioxidant properties, wherein the stem cell culture medium comprises baicalin, quercetin, liquiritin and catechins.

[0008] In the present invention, the stem cell culture medium with antioxidant properties uses a mixture of baicalin, quercetin, liquiritin and catechins as a component of the culture medium to stimulate the stem cells, thereby changing the oxidative stress state of the stem cells so that the stem cells can secrete exosomes with antioxidant effects, and the exosomes secreted by the stem cells have higher antioxidant capacity.

[0009] Baicalin is a flavonoid compound extracted from plants such as Scutellaria baicalensis, which has multiple biological activities, such as anti-inflammatory and antibacterial effects. In the present invention, as one of the components of the stem cell culture medium, it can provide a favorable microenvironment for the growth and functional maintenance of stem cells. Quercetin is a flavonol compound widely present in plants, which has antioxidant and anti-apoptotic properties and helps to regulate the physiological state of stem cells. Liquiritin is derived from licorice and has certain immunomodulatory and antioxidant abilities. It works synergistically with other ingredients to promote the normal metabolism and activity maintenance of stem cells. Catechins are one of the main active ingredients in tea, have strong antioxidant effects, can effectively scavenge free radicals, reduce the damage of oxidative stress to stem cells, and may also play a role in cell signal transduction and other aspects, jointly promoting the good growth and functional performance of stem cells in the culture medium.

[0010] Among them, the baicalin, quercetin, liquiritin and catechin selected by the present invention all have flavonoid compounds, and the functional groups such as hydroxyl and carbonyl in the structure can prevent the generation of free radicals and eliminate their antioxidant activity, thereby avoiding oxidative damage. They can act as free radical scavengers, effectively remove active oxygen free radicals such as hydrogen peroxide and hydroxyl free radicals in the body, and protect cells from damage by oxidative stress.

[0011] The selected mixture of baicalin, quercetin, liquiritin and catechin, compared with a single one or a combination of any two of them, the mixture of this scheme can further secrete exosomes with antioxidant effects on stem cells.

[0012] The corresponding culture medium may also include some basic culture medium, such as DMEM / F12 culture medium, which is used to provide basic nutrition for the culture of stem cells.

[0013] As an optional embodiment of the first aspect of the present invention, the stem cell culture medium with antioxidant properties comprises 1-20 μg / mL baicalin, 1-10 μM quercetin, 1-20 μg / mL liquiritin, and 0.1-2 μg / mL catechins.

[0014] Specifically, the concentration of baicalin added is 1 to 20 μg / mL, which specifically means that the content of baicalin in each milliliter of culture medium is between 1 μg and 20 μg. Specifically, the concentration of baicalin can be 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL.

[0015] The concentration range of quercetin is 1 to 10 μM, which specifically means that the molar concentration of quercetin per liter of culture medium is between 1 μmol and 10 μmol. Specifically, in the culture medium, the concentration of quercetin can be 1 μM, 3 μM, 5 μM, 8 μM, or 10 μM.

[0016] The added concentration of the liquiritin is 1 to 20 μg / mL, which specifically means that the content of liquiritin in each milliliter of culture medium is between 1 μg and 20 μg. The specific concentration of the liquiritin can be 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL.

[0017] The added concentration of catechins is 0.1-2 μg / mL, which specifically means that the content of catechins in each milliliter of culture medium is between 0.1 μg and 2 μg. The specific concentration of catechins can be 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, and 2 μg / mL.

[0018] This concentration range ensures the effectiveness and stability of each component in the culture medium while avoiding toxic effects on stem cells.

[0019] As an optional embodiment of the first aspect of the present invention, the stem cell culture medium includes 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL liquiritin, and 0.5 μg / mL catechins.

[0020] More preferably, at this concentration of stem cell culture medium, it can enhance the antioxidant properties of the corresponding exosomes to a greater extent while avoiding toxic effects on stem cells.

[0021] As a second aspect of the present invention, a method for preparing stem cell exosomes with antioxidant properties is provided, comprising: culturing stem cells in any culture medium described in the first aspect of the present invention, and collecting the exosomes produced by the stem cells.

[0022] In the present invention, the stem cell exosomes with antioxidant properties are obtained from stem cells cultured using the culture medium of the first invention, and in the corresponding mixed culture system, the stem cells are stimulated to separate exosomes with antioxidant properties.

[0023] As an optional embodiment of the second aspect of the present invention, the stem cells are cultured for 3-4 days. Specifically, the corresponding culture time can be determined according to the cell growth and exosome production.

[0024] As an optional embodiment of the second aspect of the present invention, the extraction of exosomes specifically also includes purification of the exosomes.

[0025] The purpose of exosome purification is to remove impurities, cell debris, etc. in the culture medium to obtain high-purity exosomes. The exosomes can be purified by differential centrifugation, ultrafiltration, or immunomagnetic beads to collect the produced exosomes.

[0026] Ultracentrifugation is a classic method that gradually increases centrifugal force to separate substances of different sizes and densities, thereby obtaining exosome precipitation. Ultrafiltration uses filter membranes with different pore sizes to screen and separate exosomes according to their size. Immunomagnetic separation is based on specific markers on the surface of exosomes, and uses the properties of magnetic beads binding to antibodies to specifically capture exosomes. Through purification, the stability and effectiveness of exosomes in subsequent applications can be improved, making their role in skin care products or medicines more accurate and reliable.

[0027] In a specific embodiment of the present invention, the method for purifying exosomes is a combination of centrifugation and ultrafiltration. Specifically, it can be: collecting the exosome supernatant after 3-4 days of culture; centrifuging the collected supernatant, filtering the supernatant, and obtaining the supernatant after filtration to obtain antioxidant synergistic exosomes.

[0028] In an optional embodiment of the second aspect of the present invention, the stem cell is selected from one of hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, neural stem cells, and induced pluripotent stem cells.

[0029] In an optional embodiment of the second aspect of the present invention, the stem cells are mesenchymal stem cells.

[0030] Mesenchymal stem cells have a wide range of sources, such as bone marrow, adipose tissue, etc. They have the advantages of being easy to obtain, having strong expansion ability, and having low immunogenicity.

[0031] In the second aspect of the present invention, the stem cell exosomes prepared by the above-mentioned preparation method have significant antioxidant properties due to the culture of stem cells in a specific culture medium. Due to the significant antioxidant properties of these exosomes, they have potential application value in intercellular tissue repair and the treatment or prevention of diseases related to antioxidant properties. For example, in skin tissue, its antioxidant properties can neutralize free radicals produced by external factors such as ultraviolet rays, reduce oxidative damage to skin cells, and delay skin aging; in vivo, it can reduce the damage of oxidative stress to organ tissues and promote the repair and regeneration of damaged tissues.

[0032] As the third aspect of the present invention, protection is sought for a stem cell exosome prepared by any method described in the second aspect of the present invention.

[0033] The stem cell exosomes of the present invention are isolated after stimulation and culture with the culture system of the first aspect of the present invention. Compared with the stem cell exosomes cultured in normal culture medium, the stem cell exosomes secreted by the exosomes have higher antioxidant capacity.

[0034] The stem cell exosomes generated after stimulation in the present invention have excellent antioxidant and anti-aging properties, and have better performance in anti-cell oxidative death test, cell apoptosis test, and anti-aging test. The stem cell exosomes of the present invention can be widely used in cell antioxidant damage, anti-aging or corresponding cell antioxidant and anti-aging.

[0035] As a fourth aspect of the present invention, protection is sought for a composition comprising stem cell exosomes prepared by any method as described in the second aspect of the present invention and an auxiliary material for use in skin care products or medicines.

[0036] In the fourth aspect of the present invention, the composition comprises the stem cell exosomes prepared by the method and auxiliary materials that can be used in the field of skin care products or medicines.

[0037] The corresponding excipients in the field of skin care products refer to excipients that can be added to skin care products, such as water, surfactants, moisturizers, etc., which can make the stem cell exosomes better dispersed in the product and maintain the stability and comfort of the product. The specific scheme is not limited. After the corresponding stem cell exosomes and skin care excipients are added to the composition, it has antioxidant damage and anti-aging effects, and can be used as an antioxidant damage and anti-aging product. The application of the composition can be a skin care product with antioxidant and / or anti-aging properties, and the skin care product can be an aqueous solution, cream, powder, oil, etc., such as skin care water, lotion, face cream, etc.

[0038] The excipients that can be used in the corresponding pharmaceutical field include pharmaceutically acceptable carriers, excipients, diluents, etc., which are compatible with the active ingredients of the drug. These excipients help to make stem cell exosomes into suitable dosage forms, such as tablets, capsules, injections, etc., to facilitate the storage, transportation and use of drugs, so that the exosomes can be accurately delivered to the target site to exert their antioxidant and therapeutic effects.

[0039] The stem cell culture medium proposed by the present invention uses a mixture of baicalin, quercetin, liquiritin and catechin as a component of the culture medium to stimulate the stem cells, changing the oxidative stress state of the stem cells so that the stem cells can secrete exosomes with antioxidant effects, and the exosomes secreted by the stem cells have higher antioxidant capacity. The corresponding stem cell exosomes perform better in antioxidant performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 The toxic effects of different concentrations of baicalin on umbilical cord mesenchymal stem cells;

[0042] Figure 2 The toxic effects of different concentrations of quercetin on umbilical cord mesenchymal stem cells;

[0043] Figure 3 Toxicity of different concentrations of liquiritin on umbilical cord mesenchymal stem cells;

[0044] Figure 4 Toxicity of different concentrations of catechins on umbilical cord mesenchymal stem cells;

[0045] Figure 5 Examples of exosome particle size (NTA) in different experimental groups;

[0046] Figure 6 Examples of transmission electron microscopy (TEM) images of exosomes in different experimental groups;

[0047] Figure 7 Examples of flow cytometry for apoptosis detection in different experimental groups;

[0048] Figure 8 Examples of β-galactosidase staining in different experimental groups. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] The present invention provides stem cell exosomes with antioxidant properties, which are produced by stem cells cultured in a specific culture medium containing a mixture of baicalin, quercetin, liquiritin and catechins. Compared with existing dedicated stem cell culture media, it has better antioxidant and anti-aging properties.

[0051] Specifically, in the embodiments of the present invention, mesenchymal stem cells are used as culture cells for verification, specifically umbilical cord mesenchymal stem cells are used.

[0052] The umbilical cord mesenchymal stem cell exosomes of the embodiment of the present invention are prepared by the following steps:

[0053] S1: Preparation of culture medium

[0054] A stem cell culture medium containing corresponding components is prepared according to a ratio, wherein the corresponding stem cell culture medium is added with a basic culture medium DMEM / F12 culture medium.

[0055] S2: Culture of umbilical cord mesenchymal stem cells

[0056] The third generation umbilical cord mesenchymal stem cells were obtained and inoculated into culture bottles, and the corresponding culture medium was added and cultured for 3 days.

[0057] In this step, the third generation umbilical cord mesenchymal stem cells are used for culture. The corresponding culture environment is a 37°C, 5% CO2 incubator. During the culture process, the mixture of baicalin, quercetin, liquiritin, and catechins will gradually affect the transcription and secretion functions of stem cells, so that the exosomes secreted by them have better antioxidant properties.

[0058] The separation and subculture method of umbilical cord mesenchymal stem cells is as follows: collect umbilical cord samples under sterile conditions; then rinse with PBS buffer containing double antibody solution, and cut them into pieces;

[0059] The chopped umbilical cord tissue pieces were evenly inoculated at the bottom of the culture bottle. DMEM was used as the basic culture medium, and 10% FBS and 1% double antibody solution were added. The inoculated culture bottle was placed in an incubator at 37° C. and 5% CO 2 for culture.

[0060] When the cell confluence of mesenchymal stem cells reaches about 70%-80%, they are passaged.

[0061] S3: Isolation and purification of exosomes

[0062] After 3 days of culture, the corresponding exosome supernatant was collected and separated and purified. In the embodiment of the present invention, the separation and purification method is as follows:

[0063] The collected exosome supernatant is centrifuged to remove microvesicles or protein aggregates, and the supernatant after centrifugation is collected and then filtered using an exosome separation and enrichment nanochip. The filtered solution is then used to obtain a stem cell exosome solution.

[0064] Specifically, it includes the following sub-steps:

[0065] S31: The collected exosome supernatant was centrifuged at 4°C, 12000 g for 30 min to remove microvesicles or protein aggregates, and the supernatant was collected.

[0066] S32: Dilute the supernatant with PBS solution.

[0067] S33: Filter the diluted supernatant with a 0.45 μm filter and take the secondary supernatant after filtration.

[0068] S34: Filter the secondary supernatant with a 0.22 μm filter to remove apoptotic bodies and microvesicles, and take the filtered supernatant.

[0069] S35: Use the exosome separation and enrichment nanochip for filtration, and the filtered solution is the stem cell exosome solution.

[0070] The following experiments are simultaneously provided for verification in the specific implementation manner of the present invention.

[0071] 1. Toxicity test of components of different concentrations on umbilical cord mesenchymal stem cells

[0072] The first aspect of the specific implementation of the present invention is used to verify the toxicity of components at different concentrations to umbilical cord mesenchymal stem cells. Specifically, in the present invention, baicalin, quercetin, liquiritin, and catechin are all flavonoids. In order to determine their toxic effects on mesenchymal stem cells, in the first aspect of the embodiment of the present invention, different concentrations of baicalin, quercetin, liquiritin, and catechin were used to culture P3 umbilical cord mesenchymal stem cells for 3 days, and then the CCK-8 kit was used to detect the cell survival at each concentration.

[0073] 1.1 Toxicity test of different concentrations of baicalin on umbilical cord mesenchymal stem cells

[0074] Baicalin at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL was used to culture P3 umbilical cord mesenchymal stem cells for 3 days, and then the CCK-8 kit was used to detect the cell survival at each concentration. The results are shown in Table 1 and Figure 1 shown.

[0075] Table 1. Effects of different concentrations of baicalin on umbilical cord mesenchymal stem cells

[0076] Group Survival rate (%) Blank control group 100.00±2.96 1μg / mL baicalin 97.93±4.91 5μg / mL baicalin 102.42±3.56 10μg / mL baicalin 100.58±8.37 20μg / mL baicalin 106.05±4.54

[0077] Note: Compared with the blank control group, there was no significant difference in the baicalin groups at each concentration, p>0.5.

[0078] From Table 1 and Figure 1 It can be seen that the addition of 1-20 μg / mL baicalin has no significant difference on umbilical cord mesenchymal stem cells. At the same time, 20 μg / mL baicalin has a certain effect on improving the survival rate of umbilical cord mesenchymal stem cells. Therefore, the concentration range of baicalin in the present invention is 1-20 μg / mL, preferably 20 μg / mL.

[0079] 1.2 Toxicity test of different concentrations of quercetin on umbilical cord mesenchymal stem cells

[0080] The P3 generation umbilical cord mesenchymal stem cells were cultured with quercetin at concentrations of 1 μM, 2.5 μM, 5 μM, and 10 μM for 3 days, and then the cell survival at each concentration was detected using a CCK-8 kit. The results are shown in Table 2 and Figure 2 shown.

[0081] Table 2. Effects of different concentrations of quercetin on umbilical cord mesenchymal stem cells

[0082] Group Survival rate (%) Blank control group 100.00±2.96 1 μM Quercetin 107.19±4.04 2.5 μM quercetin <![CDATA[127.60±4.27 a ]]> 5 μM quercetin <![CDATA[113.80±4.95 a ]]> 10 μM quercetin <![CDATA[75.64±0.85 a ]]>

[0083] Note: Compared with the blank control group, the quercetin groups at each concentration showed significant differences, p < 0.5.

[0084] From Table 2 and Figure 2 It can be seen that the effects of adding 1-10 μM quercetin on cell survival rate are different, and there are significant differences between 2.5 μM and 10 μM and the blank control group. Among them, quercetin can significantly improve the survival rate of the sample within a certain concentration range of 2.5-5 μM, while too high a concentration such as 10 μM will significantly reduce the survival rate of the sample. Therefore, in the present invention, the amount of quercetin added is preferably 2.5-5 μM, and 2.5 μM quercetin is most preferably used.

[0085] 1.3 Toxicity test of different concentrations of liquiritin on umbilical cord mesenchymal stem cells

[0086] The P3 generation umbilical cord mesenchymal stem cells were cultured with liquiritin at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL for 3 days, and then the cell survival at each concentration was detected using a CCK-8 kit. The results are shown in Table 3 and Figure 3 shown.

[0087] Table 3. Effects of different concentrations of liquiritin on toxicity of umbilical cord mesenchymal stem cells

[0088] Group Survival rate (%) Blank control group 100.00±2.96 1μg / mL Liquorice 103.43±4.05 5μg / mL Licorice glycoside <![CDATA[112.00±4.99 a ]]> 10μg / mL Liquorice 104.30±5.15 20μg / mL liquiritin <![CDATA[120.05±2.52 a ]]>

[0089] Note: Compared with the blank control group, the liquiritin groups at each concentration showed significant differences, p < 0.5.

[0090] From Table 3 and Figure 3 It can be seen that the addition of 1-20 μg / mL glycyrrhizin has a promoting effect on the survival rate of umbilical cord mesenchymal stem cells, and it is judged that with the increase of the added amount, its promoting effect on the survival of umbilical cord mesenchymal stem cells increases. Therefore, the concentration range of glycyrrhizin in the present invention is 1-20 μg / mL, and preferably 20 μg / mL.

[0091] 1.4 Toxicity test of different concentrations of catechins on umbilical cord mesenchymal stem cells

[0092] The P3 generation umbilical cord mesenchymal stem cells were cultured with catechins at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL for 3 days, and then the cell survival at each concentration was detected using a CCK-8 kit. The results are shown in Table 4 and Figure 4 shown.

[0093] Table 4. Effects of different concentrations of catechins on toxicity of umbilical cord mesenchymal stem cells

[0094] Group Survival rate (%) Blank control group 100.00±2.96 0.1μg / mL catechins 96.03±1.43 0.5μg / mL catechins <![CDATA[113.62±2.14 a ]]> 1μg / mL catechins <![CDATA[91.81±0.81 a ]]> 2μg / mL catechins <![CDATA[94.03±2.65 a ]]>

[0095] Note: Compared with the blank control group, there are significant differences in each concentration of catechins, p < 0.5.

[0096] From Table 4 and Figure 4 It can be seen that the addition of 0.1-2 μg / mL catechins has a certain effect on the survival rate of umbilical cord mesenchymal stem cells, among which 0.5 μg / mL catechins has a promoting effect on the survival of umbilical cord mesenchymal stem cells, so the addition amount of catechins is preferably 0.5 μg / mL catechins.

[0097] According to the above tests, the first aspect of the specific embodiment of the present invention determines the optimal culture concentration of each compound: 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL liquiritin, and 0.5 μg / mL catechin.

[0098] 2. Detection of exosome shape and size

[0099] As a second aspect of a specific embodiment of the present invention, the following experimental groups are provided for control testing to verify whether the exosomes extracted from the corresponding culture medium have any effect on the shape and particle size.

[0100] Experimental Group:

[0101] Experimental group A: blank control culture medium umbilical cord mesenchymal stem cell exosome group;

[0102] Experimental group B: umbilical cord mesenchymal stem cell exosomes group containing mixed culture medium containing baicalin + quercetin + liquiritin + catechin;

[0103] Experimental group C: umbilical cord mesenchymal stem cell exosomes group in baicalin + liquiritin mixed culture medium;

[0104] Experimental group D: quercetin + catechin mixed culture medium umbilical cord mesenchymal stem cell exosome group;

[0105] Among them, in each experimental group, the concentration of baicalin was 20 μg / mL, the concentration of quercetin was 2.5 μM, the concentration of liquiritin was 20 μg / mL, and the concentration of catechin was 0.5 μg / mL.

[0106] Specifically, in this embodiment, umbilical cord mesenchymal stem cells were cultured in a stem cell culture medium containing 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL licorice glycosides, and 0.5 μg / mL catechins, and a normal stem cell-specific culture medium was used as a blank control culture medium group. After 3 days of culture, the supernatant exosomes were extracted, and the particle size, distribution and morphology were detected by NTA and TEM. Among them, the extraction method is the same as the first aspect of the embodiment of the present invention, and will not be repeated here.

[0107] In the experiment of the present invention, the normal stem cell-specific culture medium corresponding to the blank control group is a commercially available culture medium, the corresponding brand is Meallcell, and the product specification is MAO 108 500 mL.

[0108] The particle size distribution of exosomes was detected by nanoparticle tracking analysis (NTA) technology, and the morphology of exosomes was observed by transmission electron microscopy (TEM). The specific test results are shown in Table 5 and Figure 5 , Figure 6 shown.

[0109] Table 5. Exosome particle size

[0110]

[0111]

[0112] From Table 5 and Figure 5 , Figure 6 It can be seen that there are differences in the particle size distribution of umbilical cord mesenchymal stem cell exosomes under different culture medium conditions, and specific culture medium components will increase the number of exosomes in a specific particle size range.

[0113] The examples of the present invention show that different culture media have certain effects on the secreted exosomes, which are specifically manifested in different particle sizes.

[0114] 3. Exosome Function Detection

[0115] As a third aspect of a specific embodiment of the present invention, the present invention provides corresponding experiments and tests for verifying exosomes with antioxidant properties.

[0116] 3.1 Anti-cell oxidative death experiment

[0117] Experimental groups: blank control group, H2O2 model group, H2O2+A group, H2O2+B group, H2O2+C group, H2O2+D group

[0118] This experiment used the H2O2-induced damage model. H2O2 has a significant damaging effect on cells and significantly reduces cell survival rate. By verifying the survival rate of cells in this model, we can verify whether the corresponding different exosome groups can improve the cell damage caused by H2O2, thereby verifying their antioxidant capacity.

[0119] Human immortalized keratinocytes (HaCat cells) in the logarithmic growth phase were plated in 96-well plates, and 5×10 3 / well, adhered to the wall and cultured for 24h. The blank control group, H2O2 model group, H2O2+A (exosome 5×10 7 Particles / mL) group, H2O2+B (exosomes 5×10 7 Particles / mL) group, H2O2+C (exosomes 5×10 7 Particles / mL) group, H2O2+D group (exosomes 5×10 7 Particles / mL) groups were divided into groups for treatment. After 24 hours, the CCK-8 kit was used to detect the cell survival rate of each group. The results are as follows:

[0120] Table 6. Relative survival rate of cells in each group (%)

[0121] Group Survival rate (%) Blank control group <![CDATA[100±1.50 a ]]> <![CDATA[H2O2 model group]]> 50.29±0.99 <![CDATA[H2O2+A(5×10 7 Particles / mL) group]]> <![CDATA[60.78±1.43 a ]]> <![CDATA[H2O2+B(5×10 7 Particles / mL) group]]> <![CDATA[79.26±1.19 a,b ]]> <![CDATA[H2O2+C(5×10 7 Particles / mL) group]]> <![CDATA[70.64±1.83 a,b,c ]]> <![CDATA[H2O2+D(5×10 7 Particles / mL) group]]> <![CDATA[64.47±2.11 a,c ]]>

[0122] Note: a indicates that compared with the model group, there is a significant difference, P < 0.05; b indicates that compared with the blank control culture medium umbilical cord mesenchymal stem cell exosome group (Group A), there is a significant difference, P < 0.05; c indicates that compared with the umbilical cord mesenchymal stem cell exosome group (Group B) of the mixed culture medium of baicalin + quercetin + licorice glycoside + catechin, there is a significant difference, P < 0.05.

[0123] It can be concluded from Table 6 that the cell survival rate in group B reached more than 79%, which was significantly higher than that in other groups, indicating that group B significantly improved the survival rate of cells after H2O2 damage at this concentration and had a more significant antioxidant property.

[0124] In combination with the second aspect of the embodiment of the present invention, it is shown that different culture medium components not only affect the particle size, but also affect its antioxidant properties. Compared with the four experimental groups, the experimental group B of the present invention, that is, the culture medium with a mixture of baicalin + quercetin + liquiritin + catechin culture medium in the embodiment of the present invention, can more effectively stimulate the corresponding stem cells to secrete exosomes with antioxidant properties.

[0125] 3.2 Cell apoptosis assay

[0126] This experiment aims to study the effects of different treatments on the survival rate of human immortalized keratinocytes (HaCat cells). By setting up a blank control group, a H2O2 model group, and a treatment group with different additives (A and B) combined with H2O2, the effects of these treatments on cell apoptosis were observed, and the effects of different treatments on cell survival were evaluated.

[0127] Human immortalized keratinocytes (HaCat cells) in the logarithmic growth phase were plated in 6-well plates, and 5×10 5 / well, adhered to the wall and cultured for 24h. The blank control group, H2O2 model group, H2O2+A (2×10 8 Particles / mL) group, H2O2+B(5×10 7 Particles / mL) group, H2O2+B(2×10 8 Particles / mL) group, and after 24 hours, the cells were stained with Annexin V / PI apoptosis detection kit, and then flow cytometry was performed. The results are shown in Figure 7 As shown in Table 7.

[0128] Table 7. Cell survival rate of each group (%)

[0129]

[0130]

[0131] Note: a indicates that there is a significant difference compared with the model group, P < 0.05; b indicates that there is a significant difference compared with group A, P < 0.05.

[0132] From Table 7 and Figure 7 It can be concluded that the concentration of exosome A used in the experiment has a poor effect on improving H2O2-induced cell damage, while the addition of exosome B can effectively improve the survival rate of cells after H2O2 damage, and the protective effect increases with increasing concentration, indicating that exosomes in group B have good application potential in improving H2O2-induced cell damage.

[0133] 3.3 Anti-aging experiment

[0134] This experiment aims to study the effects of different treatments on the aging of human immortalized keratinocytes (HaCat cells). By setting up a blank control group, a H2O2 model group, and a treatment group with different exosomes (A and B) and H2O2 combinations, β-galactosidase staining was used to detect cell aging, and the degree of cell aging was evaluated by the percentage of positive cells. The specific experimental methods are as follows:

[0135] Human immortalized keratinocytes (HaCat cells) in the logarithmic growth phase were plated in 24-well plates, and 2×10 4 / well, adhered to the wall and cultured for 24h. The blank control group, H2O2 model group, H2O2+A (exosome 5×10 7 Particles / mL) group, H2O2+B (exosomes 5×10 7 Particles / mL) group, H2O2+B(2×10 8 Particles / mL) groups were treated in groups. After 24 hours, the cells were stained with a β-galactosidase staining kit, photographed under a microscope, and the percentage of positive cells was analyzed using Image J software. The results are shown in Figure 8 And Table 8.

[0136] Table 8. Proportion of cells with positive β-galactosidase staining in each group

[0137] Group Positive cell percentage (%) Blank control group <![CDATA[0.21±0.02 a ]]> <![CDATA[H2O2 model group]]> 5.08±0.14 <![CDATA[H2O2+A(2×10 8 Particles / mL) group]]> <![CDATA[1.23±0.13 a ]]> <![CDATA[H2O2+B(5×10 7 Particles / mL) group]]> <![CDATA[0.92±0.04 a,b ]]> <![CDATA[H2O2+B(2×10 8 Particles / mL) group]]> <![CDATA[0.54±0.08 a,b ]]>

[0138] Note: a indicates that there is a significant difference compared with the model group, P < 0.05; b indicates that there is a significant difference compared with group A, P < 0.05.

[0139] Combination Figure 8 and Table 8, indicating that exosome A can alleviate H2O2-induced cell senescence to a certain extent, but the effect is not as good as exosome B. Exosome B can effectively reduce the degree of cell senescence, and with the increase of concentration, the anti-cell senescence effect is enhanced, which has potential application value in combating H2O2-induced cell senescence.

[0140] In summary, the stem cell exosomes of the present invention have improved antioxidant and anti-aging properties, and have potential in corresponding antioxidant and anti-aging application scenarios. The corresponding application scenario can be to apply the stem cell exosomes of this solution to skin care products or medicines, and the skin care products or medicines based on the stem cell exosomes have antioxidant and anti-aging effects.

[0141] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A stem cell culture medium, characterized in that The stem cell culture medium comprises baicalin, quercetin, liquiritin and catechins.

2. The stem cell culture medium according to claim 1, characterized in that The stem cell culture medium comprises 1-20 μg / mL baicalin, 1-10 μM quercetin, 1-20 μg / mL liquiritin, and 0.1-2 μg / mL catechin.

3. The stem cell culture medium according to claim 1, characterized in that The stem cell culture medium includes 20 μg / mL baicalin, 2.5 μM quercetin, 20 μg / mL liquiritin, and 0.5 μg / mL catechin.

4. A method for preparing stem cell exosomes with antioxidant properties, characterized in that: The method comprises culturing stem cells in a culture medium as described in any one of claims 1 to 3, and collecting exosomes produced by the stem cells.

5. The method for preparing stem cell exosomes with antioxidant properties according to claim 4, characterized in that: The stem cells are cultured for 3-4 days.

6. The method for preparing stem cell exosomes with antioxidant properties according to claim 4, characterized in that: Purification of exosomes is also included.

7. The method for preparing stem cell exosomes with antioxidant properties according to claim 4, characterized in that: The stem cell is selected from hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, neural stem cells, and induced pluripotent stem cells.

8. The method for preparing stem cell exosomes with antioxidant properties according to claim 4, characterized in that: The stem cells are mesenchymal stem cells.

9. A stem cell exosome prepared by the method according to any one of claims 4 to 8.

10. A composition comprising stem cell exosomes prepared by the method according to any one of claims 4 to 8 and an auxiliary material acceptable in the field of skin care products or medicines.

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