Stem cell exosome repair gel and preparation method thereof

Abstract

The invention belongs to the field of cell biology, particularly relates to stem cell exosome repair gel and a preparation method thereof, and more particularly relates to stem cell exosome repair gel containing active biological peptide and a preparation method thereof. The invention provides a repairing composition which comprises an active peptide as shown in SEQ ID NO.1 and a stem cell exosome. The repair gel provided by the invention contains the active peptide and the stem cell exosome loaded with the bioactive substances, and the components have a synergistic effect, so that the expression level of related cell factors can be improved, the repair after skin injury is promoted, and the physiological state of the skin is improved.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, and specifically, relates to a stem cell exosome repair gel and a preparation method thereof, and more specifically, relates to a stem cell exosome repair gel containing active biological peptides and a preparation method thereof. Background Art

[0002] Exosomes are a type of extracellular vesicles with a diameter of about 30-150nm secreted by cells. They carry a variety of bioactive substances from donor cells into effector cells and effectively regulate the body's cell signal transduction. Studies have shown that exosomes secreted by stem cells physiologically repair or remove damaged, diseased and aged cells by delivering bioactive substances from stem cells to effector cells, exerting anti-inflammatory, immune regulation, promoting effector cell proliferation, migration and differentiation, and promoting angiogenesis. Therefore, they are often used for skin repair and other operations.

[0003] Existing skin repair and care operations mostly use chemical substances, which may cause certain adverse effects, such as thinning of the skin barrier. Therefore, more and more attention is focused on biological substances, and stem cell exosomes, as a biological substance rich in various nutrients, are used in the treatment of skin damage. However, using only stem cell exosomes for treatment cannot achieve good results.

[0004] Therefore, there is a need in the art for a stem cell exosome and a preparation method thereof, which can achieve a good skin repair effect. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a repair composition comprising: Active peptides as shown in SEQ ID NO.1, and stem cell exosomes.

[0006] Furthermore, the sequence of the active peptide is as follows: Tyr Asp Glu Glu Val Gly Met Leu Gln Glu His Leu Met Gln Ile Ala ArgHis Glu Ile (SEQ ID NO. 1).

[0007] Furthermore, the stem cell exosomes are loaded with quercetin and tea polyphenols.

[0008] Furthermore, the stem cell exosomes are pluripotent stem cell exosomes or multipotent stem cell exosomes.

[0009] Furthermore, the stem cell exosomes are one or more of adipose stem cell exosomes, mesenchymal stem cell exosomes, and epithelial stem cell exosomes. Preferably, the stem cell exosomes are adipose stem cell exosomes.

[0010] Furthermore, the stem cells are of human origin.

[0011] Furthermore, the stem cells are derived from human umbilical cord tissue and adipose tissue. Preferably, the stem cells are derived from human adipose tissue.

[0012] Furthermore, the adipose stem cell exosomes are prepared by the following steps: 1) Obtaining human adipose tissue; 2) Enzymatic digestion of adipose tissue; 3) Obtaining adipose stem cells; 4) Cultivate adipose stem cells; 5) Ultracentrifuge adipose-derived stem cells to obtain adipose-derived stem cell exosomes.

[0013] Furthermore, the enzyme for enzymolysis is type I collagenase, type II collagenase, type IV collagenase, type V collagenase, or a mixture thereof.

[0014] Furthermore, the ultracentrifugation conditions are 4° C., 10,000 rpm, for 60-90 min.

[0015] Furthermore, the stem cell exosomes loaded with quercetin and tea polyphenols are prepared by encapsulating a certain dose of quercetin and tea polyphenols into stem cell exosomes through incubation, electroporation, extrusion, ultrasound, freeze-thaw or saponin treatment.

[0016] In some specific embodiments, the concentration of quercetin and tea polyphenols loaded in stem cell exosomes is 50-100 μg / mL.

[0017] In a specific embodiment, stem cell exosomes loaded with quercetin and tea polyphenols are obtained by co-incubation: stem cell exosomes are mixed with quercetin and tea polyphenols, and incubated at a certain temperature for a certain time. The above solution is then placed in an ultrafiltration tube with a certain molecular weight cutoff, and ultrafiltration and washing are performed three times using a biocompatible medium to obtain drug-loaded exosomes.

[0018] Furthermore, the concentration of stem cell exosomes was 1×10 6 / mL–1×10 12 / mL, preferably 1×10 10 / mL; the incubation temperature ranges from 4°C to 50°C, preferably 37°C; the incubation time ranges from 1h to 48h, preferably 4h; the molecular weight cutoff range of the ultrafiltration tube is 10KDa to 5000KDa, preferably 100KDa; the biocompatible medium is physiological saline, physiological buffer, cell culture medium, etc.

[0019] The repair gel provided by the present invention contains active peptides and stem cell exosomes loaded with bioactive substances, and these components have a synergistic effect and can increase the expression level of related cytokines, thereby promoting the repair of skin damage and improving the physiological state of the skin.

[0020] In a second aspect, the present invention provides use of the above-mentioned repair composition for preparing a skin repair agent.

[0021] Furthermore, the present invention provides use of the above-mentioned repair composition for preparing stem cell exosome repair gel.

[0022] In a third aspect, the present invention provides a stem cell exosome repair gel, comprising the composition as described above. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0024] Example 1. Preparation of adipose stem cell exosomes (1) Adipose tissue is removed by suction.

[0025] (2) The adipose tissue was pulverized by ultrasonication and large particles were removed by filtration; then a 1% type I collagenase solution was added, the mixture was stirred and shaken thoroughly, and the mixture was digested at 37°C for 1-2 hours; (3) The enzymatically hydrolyzed fat tissue is centrifuged at 5000 rpm for 5-10 min. After centrifugation, it is divided into three layers, namely, a top layer of fat, a middle layer of mixture, and a bottom layer of liquid. The bottom layer of liquid is discarded, the top layer of fat is filtered to remove, and the middle layer of fat stem cells is obtained; (4) The adipose-derived stem cells were washed with sterile saline, centrifuged at 3000 rpm for 10 min, and the precipitated cells were collected. This process was repeated three times. (5) The cells were resuspended in DMEM medium containing 10% FBS, inoculated into a cell culture flask, and an appropriate amount of DMEM medium containing 10% FBS was added. The culture medium also included KGF at a concentration of 50 mg / mL, bFGF at a concentration of 100 mg / mL, and HGF at a concentration of 20 mg / mL. The cells were cultured in a 37°C 5% CO2 incubator. The cell culture status was observed every 12 hours, and the culture medium was replaced every 2-3 days.

[0026] (6) When the cell confluence reaches 80%, subculture is performed for 3-5 generations to obtain a sufficient amount of adipose-derived stem cells; (7) Adipose-derived stem cells were inoculated into a clean culture dish, and when the cell confluence reached more than 80%, the medium was replaced with serum-free medium and cultured for 24 h. The cell supernatant was collected and centrifuged at 4°C 2000 r / min for 20 min. The supernatant was removed to remove cell debris, and the supernatant was resuspended and filtered through a 0.22 μm filter. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C 10000 r / min for 80 min to obtain the precipitate. After rinsing with PBS, the supernatant was centrifuged at 4°C 10000 r / min for 20 min, and the process was repeated 3 times. The precipitate was collected as adipose-derived stem cell exosomes and stored in a -80°C refrigerator.

[0027] Example 2: Preparation of adipose stem cell exosomes loaded with quercetin and tea polyphenols A 1 mg / mL physiological saline solution of quercetin and tea polyphenols was prepared, and then the pH of the solution was adjusted to about 6 with a 1N sodium hydroxide aqueous solution. 100 μL of the adipose stem cell exosome solution prepared in Example 1 (concentration 1.0*10 12 / mL), to which 1mL of quercetin and tea polyphenols saline solution was added. After incubation at 37°C for 1.5h, the solution was washed twice with 5mL of saline, and 200μL of liquid was retained to obtain a quercetin and tea polyphenols loaded adipose stem cell exosome solution. Subsequently, 50μL of the solution was taken, diluted with 200μL of acetonitrile, and centrifuged at 12000rad / min to remove protein precipitates.

[0028] Example 3: In vitro proliferation experiment of epithelial cells using the composition of the present invention Human epithelial cells HaCaT cells were selected as experimental subjects to study their promoting effects on cells in vitro.

[0029] HaCaT cells were inoculated in DMEM medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. HaCaT cells in good condition and in the exponential growth phase were taken, digested, counted, and inoculated into 96-well plates, with each well containing 200 μL of fresh culture medium and an inoculation concentration of 0.5×10 5The cells were placed in a 37°C, 5% CO2 incubator and cultured for 12 hours.

[0030] The experiment was designed according to the composition shown in Table 1 (the concentration of stem cell exosomes was 1×10 10 / mL) and added the above-mentioned HaCaT cells respectively.

[0031] Table 1 Example 1 Bioactive peptides of the present invention + stem cell exosomes loaded with quercetin and tea polyphenols Comparative Example 1 Bioactive peptides of the present invention + stem cell exosomes loaded with quercetin Comparative Example 2 Bioactive peptides of the present invention + stem cell exosomes loaded with tea polyphenols Comparative Example 3 Bioactive peptides of the present invention + stem cell exosomes loaded with resveratrol and tea polyphenols Comparative Example 4 Bioactive peptides of the present invention + stem cell exosomes loaded with quercetin and resveratrol Comparative Example 5 Comparative example: Bioactive peptides + stem cell exosomes loaded with quercetin and tea polyphenols Comparative Example 6 PBS The comparative example bioactive peptide substance is shown in SEQ ID NO. 2 (Asn Phe Gly Tyr Lys Leu Ser GlyThr Thr Ser Tyr Glu Leu Leu Val Pro Thr Tyr).

[0032] The cells to which the above substances were added were placed in a 37°C, 5% CO2 incubator and cultured for 24 hours.

[0033] The cell viability was determined by the CCK-8 method, 10 μL of CCK-8 solution was added to each well, incubated for 2 h at 5% CO2 and 37°C, and the absorbance was measured at a wavelength of 450 nm using an enzyme marker, and the cell proliferation capacity was calculated. The results are shown in Table 2. It can be seen from the table that the combination of the bioactive peptides of the present invention and the stem cell exosomes loaded with quercetin and tea polyphenols can achieve the best cell proliferation capacity, while the combination of the bioactive peptides of the comparative example and the stem cell exosomes loaded with quercetin and tea polyphenols, as well as the replacement of quercetin and tea polyphenols with another resveratrol commonly used in the art, is not as good as the embodiment of the present application. Therefore, it is shown that the combination of the bioactive peptides of the present invention and the stem cell exosomes loaded with quercetin and tea polyphenols has a synergistic effect.

[0034] Table 2 Group Cell proliferation ability Example 1 192% Comparative Example 1 131% Comparative Example 2 126% Comparative Example 3 152% Comparative Example 4 160% Comparative Example 5 150% Comparative Example 6 100% Example 4: Composition of the present invention for preventing and treating skin scars 8-week-old female C57BL / 6 mice were randomly divided into groups according to the experimental design in Table 1 above, with 20 mice in each group. 1 ml of 1 mg / ml bleomycin (dissolved in PBS) was subcutaneously injected into the back of the mice. After 3 hours, the composition shown in Table 1 was applied to the back skin of each group. Each group was treated for 4 consecutive weeks before being killed. The skin tissue was fixed with 4% paraformaldehyde, embedded in conventional paraffin and sectioned for pathological histological observation and immunohistochemical detection.

[0035] To evaluate the degree of fibrosis in each group, the dermis thickness was measured, and the results are shown in Table 3. As can be seen from the table, all exosome compositions can improve skin scars relative to the PBS composition, but it is obvious that the composition of the present invention has the best effect of improving skin scars.

[0036] Table 3

[0037] Example 5: Preparation of gel from the composition of the present invention (1) Place 1.5 g of gelatin powder in a beaker and slowly add 60 mL of PBS buffer at 37 °C. During the addition process, use a glass rod to slowly stir to dissolve the gelatin. (2) Heat in a 75°C water bath for 15 min to completely dissolve the gelatin. Remove from the water bath and let stand to cool to 37-40°C to obtain a gelatin solution. (3) Weigh 0.5 g of chitosan powder and add it to 30 mL of PBS buffer, stir and dissolve to obtain a chitosan solution; (4) After the gelatin solution and the chitosan solution are fully mixed, 10 mL of 100 μg / mL exosomes of the present invention and 100 mg / mL active peptide of the present invention are added, and after gentle stirring until uniformly dispersed, the volume is fixed to 100 mL to obtain a gel solution; (5) Quickly add the gel solution to a preheated 24-well plate and place it in a 37°C incubator for 30 min to obtain a gel.

[0038] Example 6: The gel of the present invention reduces the photodamage of ultraviolet rays to the skin The determination method of this embodiment is as follows: (1) Thirty-five healthy female BALB / c mice, 8 weeks old, free of specific pathogens were selected; (2) After anesthesia, hair removal was performed by applying 6% Na2S solution to the back of the mouse using cotton wool to remove hair within a range of 3 cm × 3 cm. (4) The mice were randomly divided into 7 groups and treated with the gel prepared according to the components in Table 1; (5) After 30 minutes, use 300 mJ / cm 2 Irradiate the hair removal area with UVB for 3 consecutive days; (6) After irradiation, all mice were killed and the skin at the depilated area was removed; (7) Rinse the obtained skin in pre-cooled saline, remove blood, wipe dry with filter paper, and place in a beaker; (8) Add 9 times the weight of normal saline, cut the obtained skin tissue into small tissue pieces, use a low-temperature grinding instrument, grind it thoroughly, and centrifuge it at 2000 rpm for 15 minutes in a low-temperature centrifuge; (9) The supernatant was taken to measure the MDA level in the skin tissue of each group of mice.

[0039] The results of MDA are shown in Table 4. It can be seen from the table that the gel with only PBS has the highest level of MDA, an oxidative damage marker in mice, while the levels of MDA, an oxidative damage marker, with active peptides and exosomes have decreased to a certain extent. However, the gel prepared using the active peptides and exosomes of the present invention has the lowest level of MDA, an oxidative damage marker, indicating that the composition of the present invention can reduce and / or repair light damage caused by ultraviolet rays.

[0040] Table 4

Claims

1. A repair composition, characterized in that include: Active peptides as shown in SEQ ID NO.1, and stem cell exosomes.

2. The composition according to claim 1, characterized in that The stem cell exosomes are stem cell exosomes loaded with quercetin and tea polyphenols.

3. The composition according to claim 1, characterized in that The stem cell exosomes are pluripotent stem cell exosomes or multipotent stem cell exosomes.

4. The composition according to claim 1, characterized in that The stem cell exosomes are one or more of adipose stem cell exosomes, mesenchymal stem cell exosomes, and epithelial stem cell exosomes.

5. The composition according to claim 1, characterized in that The stem cell exosomes are adipose stem cell exosomes.

6. The composition according to claim 5, characterized in that The adipose stem cell exosomes are prepared by the following steps: 1) Obtaining human adipose tissue; 2) Enzymatic digestion of adipose tissue; 3) Obtaining adipose stem cells; 4) Cultivate adipose stem cells; 5) Ultracentrifuge adipose-derived stem cells to obtain adipose-derived stem cell exosomes.

7. The composition according to claim 6, characterized in that The enzyme for enzymolysis is type I collagenase, type II collagenase, type IV collagenase, type V collagenase, or a mixture thereof.

8. The composition according to claim 2, characterized in that The stem cell exosomes loaded with quercetin and tea polyphenols are obtained by co-incubating stem cell exosomes with quercetin and tea polyphenols.

9. Use of the composition according to any one of claims 1 to 8 for preparing stem cell exosome repair gel.

10. A stem cell exosome repair gel, characterized in that: include: The composition according to any one of claims 1 to 8.

Images