A bioactive peptide and its combined use with adipose stem cell exosomes

Abstract

The present invention discloses a bioactive peptide and its use in combination with adipose-derived stem cell exosomes. The amino acid sequence of the bioactive peptide is shown in SEQ ID NO.1. The bioactive peptide of the present invention can promote epithelial cell proliferation, thereby accelerating the repair of skin damage. When used together with adipose-derived stem cell exosomes, better effects can be achieved. At the same time, the bioactive peptide has excellent antioxidant properties, can better scavenge oxygen free radicals, achieve anti-aging effects, and realize beauty and skin care effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to a bioactive peptide and an application thereof in combination with adipose stem cell exosomes. Background Art

[0002] As people age, the social environment changes, and the pressure of life increases, their skin often develops various problems. In addition, people working in offices face the problem of computer radiation, while outdoor workers face the radiation of ultraviolet rays, which can easily lead to skin aging. As people's living standards improve, people are paying more and more attention to skin problems. Therefore, there are more and more types of cosmetics on the market.

[0003] At present, in response to various skin problems, a variety of cosmetics such as whitening, wrinkle removal, and moisturizing have appeared on the market. However, the active antioxidant and whitening ingredients in most cosmetics are chemical raw materials, and excessive use can easily cause certain damage to the skin.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The first purpose of the present invention is to provide a bioactive peptide and its application in combination with adipose stem cell exosomes. The bioactive peptide can promote epithelial cell proliferation, thereby accelerating the repair of skin damage. At the same time, the bioactive peptide has excellent antioxidant properties and can better scavenge oxygen free radicals, achieving the effect of beauty and skin care.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0007] In a first aspect, the present invention provides a bioactive peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] In the present invention, there is no strict limitation on the preparation method of the above-mentioned bioactive peptides. They can be synthesized by conventional methods in the art or commissioned to be synthesized.

[0009] The second aspect of the present invention provides a use of a bioactive peptide in the preparation of a cosmetic product that promotes epithelial cell proliferation or resists oxidation.

[0010] Preferably, the cosmetic products include skin patches, facial masks, skin creams, repair lotions and skin care lotions.

[0011] A fourth aspect of the present invention provides a skin patch for wound repair, wherein the skin patch comprises the bioactive peptide.

[0012] Preferably, the skin patch further comprises adipose stem cell exosomes.

[0013] A fifth aspect of the present invention provides a facial mask liquid, which includes the bioactive peptide.

[0014] Preferably, the facial mask liquid also includes adipose stem cell exosomes.

[0015] Preferably, the content of bioactive peptides in the facial mask liquid is 50-80 mg / ml; the content of adipose stem cell exosomes is 50-150 mg / ml.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least:

[0017] The bioactive peptide of the present invention can promote epithelial cell proliferation, thereby accelerating the repair of skin damage. When used together with adipose stem cell exosomes, it can achieve better effects. At the same time, the bioactive peptide has excellent antioxidant properties and can better scavenge oxygen free radicals, achieving anti-aging effects and achieving beauty and skin care effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0019] Figure 1 These are the results of the in vitro expansion experiment of epithelial cells in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.

[0021] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0022] An embodiment of the present invention provides a bioactive peptide, the amino acid sequence of which is shown in SEQ ID NO. 1, specifically glsalgvvtsm.

[0023] In the present invention, the preparation method of the above-mentioned bioactive peptides is not strictly limited and can be synthesized by conventional methods in the art. The bioactive peptides in the embodiments of the present invention are artificially synthesized and were accidentally found to have excellent antioxidant effects. Further experiments have shown that they have the effect of promoting epithelial cell proliferation.

[0024] The embodiments of the present invention also provide a use of a bioactive peptide in the preparation of a cosmetic product that promotes epithelial cell proliferation or resists oxidation.

[0025] In the present invention, the above-mentioned beauty products are not strictly limited. For example, they can be conventional beauty products in the field, or they can be skin patches, facial masks, skin creams, repair lotions and skin care lotions.

[0026] The bioactive peptides of the present invention can promote the proliferation of epithelial cells, thereby facilitating the repair of skin damage; therefore, another embodiment of the present invention provides a skin patch for wound repair, the skin patch comprising the bioactive peptides.

[0027] In another embodiment, the skin patch may also include exosomes derived from adipose-derived stem cells. The addition of exosomes can further promote epithelial cell proliferation. Furthermore, exosomes may help regulate immune responses and provide cells with sufficient nutrients, thereby promoting skin tissue repair.

[0028] An embodiment of the present invention further provides a facial mask liquid, which includes the above-mentioned bioactive peptide.

[0029] In addition to promoting epithelial cell proliferation, the bioactive peptide of the present invention also has excellent antioxidant properties and can effectively remove oxygen free radicals. Therefore, applying it to facial mask liquid can alleviate skin aging, prevent external environmental radiation from damaging the skin, and ultimately achieve the effect of beautifying and nourishing the skin.

[0030] To further enhance the cosmetic effect, in one embodiment, the facial mask also includes exosomes derived from adipose-derived stem cells. The addition of exosomes from adipose-derived stem cells can provide sufficient nutrients to the skin tissue while also enhancing the antioxidant effect, resulting in a better cosmetic effect.

[0031] In some embodiments, the content of bioactive peptides in the facial mask liquid can be any value between 50 and 80 mg / ml; the content of adipose stem cell exosomes can be any value between 50 and 150 mg / ml.

[0032] The technical solution of the present invention is further described in detail below through specific embodiments.

[0033] Example 1

[0034] This embodiment is a method for preparing exosomes from adipose-derived stem cells, which includes the following steps:

[0035] 1. Primary Culture of Adipose-Derived Stem Cells

[0036] 1.1 Environmental requirements: The experimental environment should be a Class 10,000 or Class 1,000 laminar flow chamber, and open operations must be performed in a Class 100 clean bench;

[0037] 1.2 Fat washing: In a clean bench, transfer the fat into a centrifuge tube containing sterile saline. Each tube should not exceed 20ml. Soak and wash thoroughly to remove any residual blood, then transfer the fat into another sterile centrifuge tube.

[0038] 1.3 Use scissors to cut the fat into about 1mm 3 Transfer small pieces to a 120ml measuring cup. Add 20ml of fat to each measuring cup;

[0039] 1.4 Add collagenase type I (1 mg / ml) in a 1:1 volume ratio, mix well, and place in a 37°C constant temperature shaker for 1 hour;

[0040] 2.5 After complete digestion, filter the larger tissue pieces using a disposable blood transfusion device, transfer the filtered liquid to a centrifuge tube, and centrifuge at 2000 rpm for 10 minutes.

[0041] 1.6 After centrifugation, discard the supernatant; mix the cells at the bottom with physiological saline, dilute to 40 ml and keep the sample for counting; centrifuge at 1000 rpm for 6 minutes;

[0042] 1.7 After centrifugation, discard the supernatant; resuspend and mix the cells in 10 ml of medium No. 13 (DMEM medium + 10% stem cell supplement), and inoculate them into a T75 culture flask.

[0043] 1.8 Cover the lid, affix the barcode, mark the culture date, generation, bottle number, etc., place it in the incubator, maintain the incubator at 37°C, saturated temperature, and CO2 concentration of 5% to start cultivation.

[0044] 2. Change the medium:

[0045] 2.1 Do not move the culture flask within 24 hours after primary cells are seeded to avoid affecting their adhesion to the wall. Observe the cells regularly afterwards.

[0046] 2.2 Choose the time for the first medium change based on the cell growth and the condition of the culture medium (generally, primary cells appear 2-3 days after seeding the bottle, the cells begin to precipitate and adhere to the wall, and the culture medium color changes from pink to light yellow, then the medium can be changed);

[0047] 2.3 Pour the culture medium in the culture flask into the waste tank and add 10ml of fresh culture medium to each culture flask;

[0048] 2.4 Place the culture bottle in the incubator for culture;

[0049] 3. Passaging: P0 to P1 (3-5 days after medium change)

[0050] 3.1 Trypsin digestion: When the cells cover about 90% of the bottom area of ​​the culture flask, trypsin digestion and subculture are performed.

[0051] 3.1.1 Aspirate the culture medium with a pipette and discard it;

[0052] 3.1.2 After gently rinsing the cells with normal saline, add 1 ml each of normal saline and 0.25% trypsin. Lay the culture flask flat and gently shake it to spread the trypsin evenly on the bottom of the flask.

[0053] 3.1.3 Observe under an inverted microscope while gently tapping the flask wall. When most cells begin to detach from the flask wall, terminate the digestion with complete culture medium.

[0054] 3.1.4 After digestion, transfer the cell suspension into a 50ml centrifuge tube. Wash the culture flask with a small amount of physiological saline and transfer the suspension into the centrifuge tube.

[0055] 3.2 Vaccination:

[0056] 3.2.1 After centrifugation, discard the supernatant, add a small amount of culture medium, and mix the cells;

[0057] 3.2.2 Subculture at a ratio of 1:2. Add 10 ml of medium 13 to each bottle, marked as P1, and indicate the subculture time and barcode.

[0058] 3.2.3 Cell expansion to 2T755;

[0059] 4 passages: P1 to P2;

[0060] 4.1 Collect the old culture medium into a 50ml centrifuge tube.

[0061] 4.2 Collect cells by trypsin digestion and centrifuge at 1000 rpm for 6 min.

[0062] 4.3 Collect 20 ml of the supernatant into the centrifuge tube in 5.1 for sample sterility testing.

[0063] 4.4 Discard the supernatant, resuspend the cells in 5 ml of culture medium, inoculate two T175 cells, and add culture medium to 18 ml per bottle;

[0064] 5 passages: P2 to P3

[0065] 5.1 Trypsin digestion: When the cells cover about 90% of the bottom area of ​​the culture flask, trypsin digestion and passage are performed;

[0066] 5.1.1 Aspirate the culture medium with a pipette and discard it;

[0067] 5.1.2 After gently rinsing the cells with normal saline, add 2.5 ml each of normal saline and 0.25% trypsin. Lay the culture flask flat and gently shake it to spread the trypsin evenly on the bottom of the flask.

[0068] 5.1.3 Observe under an inverted microscope while gently tapping the flask wall. When most cells begin to detach from the flask wall, terminate the digestion with complete culture medium.

[0069] 5.1.4 After digestion, transfer the cell suspension into a 50ml centrifuge tube. Wash the culture flask with a small amount of physiological saline and transfer the suspension into the centrifuge tube.

[0070] 5.1.5 Discard the supernatant, combine the cell pellet into a centrifuge tube, adjust the volume to 40 ml, take a sample and count; then centrifuge again at 1000 rpm for 6 minutes;

[0071] 5.2 Discard the supernatant, resuspend the cells in 6 ml of medium 13, inoculate into 6 T175 cells, and replenish the medium to 18 ml per bottle;

[0072] 5.3 Two days later, when the cells were about 90% confluent, the cell supernatant was collected for exosome extraction;

[0073] 5.4 Centrifuge the supernatant at 4000 × g to remove cells and cell debris;

[0074] 5.5 Add exosome precipitation reagent to the supernatant, mix thoroughly, let it stand, and then centrifuge at 10,000 × g for 30 minutes to collect the precipitate;

[0075] 5.6 Resuspend the precipitate in PBS and place it in a purification column. Place the purification column in a collection tube. Centrifuge the collection tube at 4000×g for 10 minutes at 4°C. Freeze-dry the solution in the collection tube to obtain human adipose-derived mesenchymal stem cell exosomes.

[0076] Example 2

[0077] This example is a study on the effects of bioactive peptides and adipose-derived stem cell exosomes on epithelial cell proliferation in vitro:

[0078] Epithelial cells were HaCaT cells (purchased from Shanghai Jining Biotechnology Co., Ltd.);

[0079] HaCaT cells were inoculated into culture medium and cultured at 37°C under 5% CO2. HaCaT cells in the logarithmic growth phase were obtained and counted.

[0080] HaCaT cells were seeded in 96-well plates (4 × 10 3 / well), add 200 μl of cell-free blank culture medium as the blank group; after culturing at 37°C, 5% CO2 for 12 hours, discard the culture medium, and add 200 μl of culture medium containing bioactive peptide rec, adipose stem cell exosomes, bioactive peptide and adipose stem cell exosomes respectively (bioactive peptide content is 60 mg / ml; adipose stem cell exosome content is 100 mg / ml); set up 6 parallel wells, add the same amount of culture medium to the negative control group, continue to culture for 24 hours, add 20 μl of MTT (5 mg / ml) to each well, incubate for 4 hours; discard the culture medium, add 150 μl of DMSO, and measure the absorbance of each well at a wavelength of 490 nm; then calculate the cell proliferation rate, cell proliferation rate = (absorbance of experimental group - absorbance of blank group) (absorbance of negative control group - absorbance of blank group) - 1;

[0081] The results are as follows Figure 1 As shown in the figure, compared with the active peptide group, **P < 0.01; Figure 1 It can be seen that the bioactive peptides in the present invention can promote the proliferation of epithelial cells in vitro, and by adding the bioactive peptides and exosomes together, the proliferation of epithelial cells can be better promoted.

[0082] Example 3

[0083] This example is a study on the antioxidant properties of bioactive peptides:

[0084] DPPH scavenging ability was determined as follows:

[0085] A 60 mg / ml aqueous solution of the bioactive peptide was prepared; 2 ml of the aqueous solution of the bioactive peptide was added; 2 ml of a 0.1 mmol / L DPPH ethanol solution was mixed, and the mixture was kept in the dark for 30 min. The OD value was measured at 517 nm, which was recorded as A; similarly, 2.0 ml of distilled water was added to 2.0 ml of a 0.1 mmol / L DPPH ethanol solution, and the OD value was measured, which was recorded as B; 2.0 ml of the aqueous solution of the bioactive peptide was added to 2.0 ml of ethanol, and the OD value was measured, which was recorded as C; the DPPH scavenging ability was calculated according to the scavenging rate = [1-(AC) / B] × 100%; the calculation results are shown in Table 1;

[0086] The hydroxyl radical scavenging ability was determined as follows:

[0087] The reaction system was incubated in a 37°C water bath for 0.5 h. The OD value was measured at 510 nm using distilled water as a reference and recorded as A. In the control group, 2.0 ml of distilled water was added instead of the 60 mg / ml bioactive peptide aqueous solution, and the OD value was measured at 510 nm and recorded as B. The hydroxyl radical scavenging ability was calculated according to the scavenging rate = (BA) / B × 100%. The calculation results are shown in Table 1.

[0088] Table 1 Antioxidant properties of bioactive peptides

[0089] Group DPPH clearance rate Hydroxyl radical scavenging rate Bioactive peptides 46.58±0.126 82.31±0.547

[0090] From Table 1 we can see that:

[0091] The bioactive peptide of the present invention has excellent antioxidant properties and can play an antioxidant role.

[0092] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A bioactive peptide, characterized in that The amino acid sequence of the bioactive peptide is shown in SEQ ID NO.

1.

2. Use of the bioactive peptide according to claim 1 in the preparation of cosmetic products that promote epithelial cell proliferation or provide anti-oxidation.

3. The use according to claim 2, characterized in that The cosmetic products include skin patches, facial masks, skin creams, repair lotions and skin care lotions.

4. A skin patch for wound repair, characterized in that: The invention comprises the bioactive peptide described in claim 1.

5. The skin patch according to claim 4, characterized in that: The skin patch also includes adipose stem cell exosomes.

6. A facial mask liquid, characterized in that: The facial mask liquid comprises the bioactive peptide according to claim 1.

7. The facial mask liquid according to claim 6, characterized in that The facial mask liquid also includes adipose stem cell exosomes.

8. The facial mask liquid according to claim 6, characterized in that The content of bioactive peptides in the facial mask liquid is 50-80 mg / ml; the content of adipose stem cell exosomes is 50-150 mg / ml.

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