CN113855619A

CN113855619A - Application of placenta stem cell exosome in preparation of skin beauty cosmetics or medicines

Info

Publication number: CN113855619A
Application number: CN202111375496.7A
Authority: CN
Inventors: 张冬久; 钟春秀
Original assignee: Beijing Yuanshengda Biotechnology Development Co ltd
Current assignee: Anling Shanghai Biotechnology Co ltd; Shandong Jiekai Biotechnology Co ltd
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2021-12-31
Anticipated expiration: 2041-11-19
Also published as: CN113855619B

Abstract

The invention relates to an application of a placental stem cell exosome in preparation of skin beauty cosmetics or medicines. The invention provides a method for efficiently promoting the secretion of exosomes of placental stem cells to prepare high-activity exosomes, and after the exosomes are introduced with active peptides, the method can remarkably promote the proliferation of fibroblasts and the repair of skin injury, can restore and maintain the elasticity and luster of skin in a young state by promoting the proliferation of the fibroblasts, prevents wrinkles and has a good application prospect.

Description

Application of placenta stem cell exosome in preparation of skin beauty cosmetics or medicines

Technical Field

The invention relates to the field of cosmetics and medicines, in particular to application of a placental stem cell exosome in preparation of skin beauty cosmetics or medicines.

Background

With the improvement of living standard and the improvement of science and technology, the life of human beings is continuously increased, and the aging phenomenon is more and more serious. The skin is used as an external organ of a human body, is in direct contact with the environment, is more easily affected by external invasion and injury, and causes aging phenomena such as dryness, wrinkles, sagging, color spots and the like. Skin aging has a range of causes including intrinsic cell proliferation and loss of repair capacity, and external environmental damage. With the continuous updating of research means, researchers have more clear understanding of the action mechanism of skin aging, including how physiological metabolism changes caused by aging result in the change of skin structure and how external stimuli cause skin damage. On the basis, the anti-aging active substances can be developed more specifically, and the anti-aging active substances can specifically inhibit or block certain aging reaction process, so that the targeted anti-aging effect is realized.

The skin can generate free radicals due to factors such as self metabolism and ultraviolet radiation, and the like, and oxidation stress is formed. Normally, the skin produces natural antioxidants to prevent active oxygen from damaging cells, but when the skin's own antioxidant capacity is insufficient to resist active oxygen stress, excessive active oxygen stress can cause cell damage, such as degradation of proteins, lipids, and even DNA, causing the skin to lose its elasticity and wrinkles. In order to balance the oxidation stress, it is very effective to supplement antioxidant substances to the skin from the outside to the inside and to stimulate the generation of antioxidant substances per se. In addition to direct damage to cells, reactive oxygen stress can also stimulate inflammatory factors in the body, causing cell aging and damage from inflammatory pathways. On one hand, the inflammatory factors can reduce the synthesis of collagen which is a precursor for synthesizing the collagen, cause structural abnormality and damage of the collagen and reduce the content of the collagen; on the other hand, the degradation of the extracellular matrix is realized by maintaining high content of matrix metalloprotease through increasing the synthesis of the matrix metalloprotease, improving the enzyme activity and promoting the inactivation of the inhibitor. Although it is known that the decline and loss of cell proliferation ability are important intrinsic factors of skin aging, how to increase the cell proliferation ability is also an emerging field that gradually starts to be initiated in recent years with the intensive research of stem cells. In the research process, the growth factor has great effects on skin recombination, repair and reconstruction, can promote the proliferation of keratinocytes and fibroblasts, can also promote the growth of other cells with reduced proliferation activity caused by aging, and can increase the thickness of epidermis.

In recent years, stem cell therapy has gradually become a new approach to repair skin damage. Research shows that the MSCs can promote skin wound surface to heal epidermal cells and fibroblasts, play an important role in the skin wound repair process, and participate in a plurality of physiological processes of wound repair, such as activation, proliferation, re-epithelialization, wound contraction, tissue reconstruction, extracellular matrix deposition and the like. In vitro studies have shown that stem cells can be directly differentiated into cell lines required for skin wound repair under the influence of local microenvironment, such as epidermal cell lines, fibroblast cell lines and endothelial cell lines, and simultaneously stem cells can secrete growth factors to promote proliferation and migration of epidermal cells, fibroblast cells and endothelial cells and further promote skin wound healing. The placenta mesenchymal stem cells are rich in sources and easy to collect, can be differentiated into mesoderm, ectoderm and endoderm cells under specific induction conditions, are easy to culture, induce and amplify in vitro, do not have immunological rejection, do not relate to ethical problems and the like in clinical application, and make great progress in animal experiments and clinical application. Stem cells can secrete a vesicle, an exosome, which is an important vector for cell-cell interaction, and when the exosome is delivered to an aging individual, the exosome can be phagocytized, and the exosome has a plurality of functional proteins which can change the aging cell. Clinical tests show that the stem cell exosome has the functions of anti-aging and repairing, and related skin care products are on the market at present.

The polypeptide can promote the secretion of collagen, and has good effect on eliminating wrinkles such as eye lines and the like corresponding to skin care products. Currently, active biological polypeptide has the advantages of safety, stability, easy water solubility, easy absorption, small molecular weight and the like, simultaneously has various biological activities of free radical removal, oxidation resistance, aging resistance and the like, can promote the proliferation of skin cells, can provide nutrition for skin, delay skin aging and promote skin wound repair, and is widely applied to skin cosmetics and anti-aging cosmetics. However, the polypeptide also has the problems of poor stability, poor cell penetrating power, poor accumulation in tissues and the like,

in order to maintain a youthful skin state, more and more consumers begin to use cosmetics having anti-aging effects, but the use of exosomes together with active peptides for anti-aging skin treatment drugs or cosmetics is now under way.

Disclosure of Invention

The invention relates to a placental stem cell exosome for improving skin physiological characteristics.

In one embodiment of the invention, the stem cell exosomes are 1 × 10⁴-1×10⁶Day exosomes are present.

In another aspect, the present invention provides a method for preparing a placental stem cell exosome. The method specifically comprises the following steps:

separating the placenta mesenchymal stem cells: shearing tissue with the thickness of 1-2 cm on the placenta villus membrane surface, shearing the tissue into 1mm multiplied by 1mm fragments, rinsing the fragments to be colorless by PBS, digesting the fragments by 0.25% trypsin and 0.2% collagenase IV37 ℃ in water bath for 1h, standing the fragments after oscillation for 1min to naturally divide the liquid into three layers, sucking the suspension of the middle layer into a centrifuge tube, adding PBS, shaking the suspension evenly, centrifuging the suspension for 5min at 700r/min, removing the supernatant, adding 3mL mesenchymal dry cell culture solution (87% DMEM, 10% Fetal Bovine Serum (FBS), 1% L-glutamine and 1% streptomycin) for precipitation, and centrifuging the suspension for 5min at 700r/min to remove the supernatant. Adding appropriate amount of culture solution, mixing, transferring into cell culture flask, and culturing at 37 deg.C with 5% CO₂And when the cultured cells grow to 80% in the incubator, carrying out trypsinization passage. During passage, digesting with 0.25% trypsin for 3-4 min, adding cell culture solution to stop digestion, centrifuging at 700r/min for 10min, discarding the digestion solution, adding cell culture solution, blowing, mixing, collecting cell suspension, inoculating into cell culture bottle, placing at 37 deg.C and 5% CO₂The culture solution in the incubator: 87% DMEM, 10% Fetal Bovine Serum (FBS), 1% L-glutamine, 1% streptomycin and the like.

Collecting prepared placenta stem cells, culturing in exosome secretion promoting culture medium (15% AGS, 0.5% ginsenoside and 84.5% DMEM/F12) for 60 hr when the cells are fused and grown to 85%, and collecting cell supernatant. Centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 10min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, filtering with 0.22 μm needle filter, and removing microbubbles and possible apoptotic bodies. Transfer the supernatant to an ultracentrifuge tube with a 20mL empty needle, 10%⁶Centrifuging at x g for 70min, removing supernatant, collecting precipitate to obtain crude extract exosome, 10%⁶Xg centrifugation for 70min, supernatant removed, 100. mu.L PBS dissolved precipitation, obtained relatively pure exosomes.

In another aspect of the present invention, there is provided a polypeptide for promoting skin injury repair, wherein the polypeptide can promote human fibroblast proliferation and simultaneously has a function of promoting skin wound repair. It is well known in the art that it promotes fibroblast proliferation, restores and maintains the elasticity and luster of skin in a young state, and reduces the occurrence of wrinkles.

The invention also provides an exosome introduced with polypeptide, in particular to an exosome dissolved in PBS buffer solution containing 50mM trehalose; converting SEQ ID NO: 1, adding the active peptide into the buffer solution, uniformly mixing, and transferring into an electric rotating cup for electric rotation, wherein the electric rotating cup is used for electric rotation under the conditions of 400V voltage, 175 muF capacitance and 41Tim capacitance. Then filtering with an inverted centrifugal ultrafiltration membrane to remove free and non-specifically bound polypeptide, thus obtaining the exosome introduced with the polypeptide.

In one embodiment of the present invention, the polypeptide-introduced stem cell exosome has an atopic skin injury repair-improving effect, and can be prepared into a cosmetic, a pharmaceutical, or a medicament.

In one embodiment of the present invention, the cosmetic is any one of a skin lotion, a skin softener, a lotion, an emulsion, a moisturizing lotion, a nutritional lotion, a massage cream, a nutritional cream, a cream moisturizer, a hand cream, a foundation cream, an essence, a nutritional essence, a package, a soap, a cleansing foam, a cleansing liquid, a cleansing cream, a body lotion, a body cleanser, a facial cleanser, a therapeutic agent, a beauty lotion, a packaged cosmetic, an ointment, a gel, a liniment, a solution, a patch, and a spray preparation.

In addition, the present invention provides a stem cell-derived exosome comprising a pharmaceutical composition for preventing or treating skin injury repair as an active ingredient.

In one embodiment of the present invention, the composition for skin damage is a cosmetic composition or may be an external preparation of the present invention. For example, the cosmetic composition may be a cream or lotion.

In one embodiment of the present invention, the composition for skin injury repair, wherein the exosomes of the stem cells are capable of promoting rapid repair of skin injury.

On the other hand, in one embodiment of the present invention, the skin external composition is used for skin damage repair and/or in the case of a cosmetic composition, the effect of the present invention is generally within a range that does not adversely affect the cosmetic or skin external preparation of the ingredient, for example, moisturizer, antioxidant, oily component, ultraviolet absorber, emulsifier, surfactant, thickener, alcohol, powder component, colorant, aqueous component, water, various skin nutrients, and the like may be appropriately mixed according to the needs of the art.

In addition, the cosmetic composition in one embodiment of the present invention and/or the skin external preparation has an effect (improvement of skin condition, etc.) that does not impair a commonly used skin improving agent and/or can be used in combination. For example, the exosome hydrogel derived from the stem cells of the present invention, hyaluronic acid, a hyaluronate salt (e.g., sodium hyaluronate, etc.), or at least one substance supported on the hyaluronic acid gel may be the same or different. In the external preparation for skin in one embodiment of the present invention and/or in the cosmetic composition, but not limited to the type of hydrogel, the gel polymer is preferably a polyol obtained by dispersing the hydrogel of the present invention. The gelling polymer is Pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum and at least one selected from 1 and 2 substances, wherein the polyhydric alcohol comprises ethylene glycol, propylene glycol, 1, 3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol and glycerol, and at least one selected from 1 substance may also be present.

In one embodiment of the present invention, the cosmetic composition and/or the external preparation for skin may include a commonly used external preparation for skin and/or cosmetic ingredients including, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, such as conventional adjuvants and carriers. Further, the skin external preparation and/or the cosmetic composition, the other components of the cosmetic composition or the skin external preparation and/or the intended use and kind in each preparation may be appropriately selected according to those skilled in the art without any difficulty.

Advantageous effects

The invention provides a method for efficiently promoting the secretion of exosomes of placental stem cells to prepare high-activity exosomes, and after the exosomes are introduced with active peptides, the method can remarkably promote the proliferation of fibroblasts and the repair of skin injury, can restore and maintain the elasticity and luster of skin in a young state by promoting the proliferation of the fibroblasts, prevents wrinkles and has a good application prospect.

Drawings

FIG. 1280 nm ultraviolet absorption results

FIG. 2 wound area results plot

FIG. 3 is a graph showing the results of TNF-. alpha.expression

Detailed Description

The technical scheme of the invention is described by combining specific embodiments. The experimental materials not particularly emphasized in the following examples are all conventional experimental materials, and are not particularly required, and are all conventional materials readily available to those skilled in the art.

Example 1 preparation of placental stem cells

Separating the placenta mesenchymal stem cells: shearing tissue with the thickness of 1-2 cm on the placenta villus membrane surface, shearing the tissue into 1mm multiplied by 1mm fragments, rinsing the fragments to be colorless by PBS, digesting the fragments by 0.25% trypsin and 0.2% collagenase IV37 ℃ in water bath for 1h, standing the fragments after oscillation for 1min to naturally divide the liquid into three layers, sucking the suspension of the middle layer into a centrifuge tube, adding PBS, shaking the suspension evenly, centrifuging the suspension for 5min at 700r/min, removing the supernatant, adding 3mL mesenchymal dry cell culture solution (87% DMEM, 10% Fetal Bovine Serum (FBS), 1% L-glutamine and 1% streptomycin) for precipitation, and centrifuging the suspension for 5min at 700r/min to remove the supernatant. Adding appropriate amount of culture solution, mixing, transferring into cell culture flask, and culturing at 37 deg.C with 5% CO₂And when the cultured cells grow to 80% in the incubator, carrying out trypsinization passage. During passage, digesting with 0.25% trypsin for 3-4 min, adding cell culture solution to stop digestion, centrifuging at 700r/min for 10min, discarding the digestion solution, adding cell culture solution, blowing, mixing uniformly, collecting cell suspension, inoculating to cell cultureBottle, standing at 37 deg.C and 5% CO₂The culture solution in the incubator: 87% DMEM, 10% Fetal Bovine Serum (FBS), 1% L-glutamine, 1% streptomycin and the like.

Identification of stem cell specific antigen of placenta mesenchymal cell, selecting placenta mesenchymal cell of third generation in exponential growth phase, digesting with 0.25% trypsin, collecting cell suspension in centrifugal tube, and adjusting cell concentration to 1 × 10⁵Mu L; taking 6 test tubes, respectively adding 15 mu L of mouse anti-human monoclonal antibodies CD29-PE, CD44-FITC, CD105-FITC, CD34-FITC and CD106-FITC into each test tube, taking mouse anti-human IgG2a-FITC and IgG1-PE as negative controls, respectively adding 150 mu L of cell suspension, uniformly mixing, standing at room temperature in a dark place for 10min, then washing for 2 times by PBS, detecting by a flow cytometer, and obtaining and analyzing by Cellquest software. The result shows that the third generation of the placental mesenchymal stem cells has 99.2 percent of CD44 positive rate, 99.3 percent of CD29 positive rate and 99.0 percent of CD105 positive rate, and does not express CD34 and CD106 (less than 1 percent), which indicates that the placental mesenchymal stem cells are obtained by separation and preparation.

Example 2 preparation of high Activity placental mesenchymal Stem cell exosomes

The placental stem cells prepared in example 1 were collected, cultured in an exosome secretion-promoting medium (15% AGS, 0.5% total ginsenosides plus 84.5% DMEM/F12) for 60 hours when the cells fused and grown to 85%, and the cell supernatant was collected. Centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 10min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, filtering with 0.22 μm needle filter, and removing microbubbles and possible apoptotic bodies. Transfer the supernatant to an ultracentrifuge tube with a 20mL empty needle, 10%⁶Centrifuging at x g for 70min, removing supernatant, collecting precipitate to obtain crude extract exosome, 10%⁶Xg centrifugation for 70min, supernatant removed, 100. mu.L PBS dissolved precipitation, obtained relatively pure exosomes.

Taking 5 mu L of exosome solution, and measuring the protein concentration of exosome by using a BCA method; dropping 1 drop of exosome on a copper mesh, carrying out negative staining on phosphotungstic acid with the volume fraction of 1%, drying at room temperature, observing the appearance of the exosome by using a transmission electron microscope, and detecting the diameter of the exosome; mu.L of the exosome solution was taken, diluted to 200. mu.L with PBS, and the diameter distribution was examined with a particle sizer. The result shows that the appearance of the exosome is cup-shaped, the exosome has a membrane structure, the diameter is distributed at (75-110) nm, and the BCA protein concentration measurement result is 2.4 g/L.

Example 3 preparation of placental mesenchymal stem cell exosomes control example

The placental stem cells prepared in example 1 were harvested, cultured in exosome-secretagogue medium (15% AGS plus 85% DMEM/F12) for 60h when the cells were confluent and grown to 85%, and the cell supernatant was collected. Centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 10min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, filtering with 0.22 μm needle filter, and removing microbubbles and possible apoptotic bodies. Transfer the supernatant to an ultracentrifuge tube with a 20mL empty needle, 10%⁶Centrifuging at x g for 70min, removing supernatant, collecting precipitate to obtain crude extract exosome, 10%⁶Xg centrifugation for 70min, supernatant removed, 100. mu.L PBS dissolved precipitation, obtained relatively pure exosomes.

Taking 5 mu L of exosome solution, and measuring the protein concentration of exosome by using a BCA method; dropping 1 drop of exosome on a copper mesh, carrying out negative staining on phosphotungstic acid with the volume fraction of 1%, drying at room temperature, observing the appearance of the exosome by using a transmission electron microscope, and detecting the diameter of the exosome; mu.L of the exosome solution was taken, diluted to 200. mu.L with PBS, and the diameter distribution was examined with a particle sizer. The result shows that the appearance of the exosome is cup-shaped, the exosome has a membrane structure, the diameter is distributed at (70-100) nm, and the BCA protein concentration measurement result is 1.3 g/L.

It can be seen from the results of examples 2 and 3 that the secretion of exosomes and the diameter distribution effect of exosomes can be remarkably promoted by adding the total ginsenoside, wherein the secretion amount of exosomes is increased by nearly 90%, and the promoting effect is good.

Example 4 exosome preparation of fusion polypeptides

Will be 4X 10⁹The exosome of (4) was dissolved in 500 μ L of PBS buffer containing 50mM trehalose; 1mg of the peptide of SEQ ID NO: 1 activity ofThe peptide is also added into the buffer solution, and is evenly mixed and transferred into an electric rotating cup for electric rotation, wherein the electric rotating cup is used for electric rotation under the conditions of voltage 400V, capacitance 175 muF and 41 Tim. The membrane was then filtered through an inverted centrifugal ultrafiltration membrane to remove free and non-specifically bound polypeptides.

Whether the polypeptide was successfully transfected was determined by ultraviolet absorption at 280nm, blank: 400 μ l PBS buffer containing 50mM trehalose; negative controls no polypeptide was added as a control: will be 4X 10⁹The exosome of (4) was dissolved in 500 μ L of PBS buffer containing 50mM trehalose; the processing flow is the same as the electrotransformation step. The ultraviolet absorption at 280nm was measured, and the test results are shown in fig. 1, and it can be seen from fig. 1 that after transduction by electroporation and elution of polypeptides not introduced into exosomes, the ultraviolet absorption value at 280nm of the experimental sample reached 0.23, which is higher than the ultraviolet absorption values of the control two groups, demonstrating that polypeptides were efficiently introduced into exosomes by electroporation.

Example 5 fibroblast proliferation promotion assay

The cell proliferation capacity was measured by MTT method by subjecting HSF in logarithmic growth phase to 1X 10³Inoculating the cells to a 96-well cell culture plate at a cell density of/mL, and replacing the cells with the cells at a concentration of 10 after the cells are completely attached to the wall⁵mL of exosomes prepared in examples 2-4 or SEQ ID NO: 1 (50ug/mL) in serum-free RPMI-1640 medium for 24 h. The normal serum-free RPMI-1640 culture solution is used as a control group, the quercetin is used as a positive control (the dosage is 50ug/mL), and each group is provided with 6 multiple wells. Mu.l of 5mg/ml MTT solution was added to each well and incubation was continued for 4h, the supernatant was discarded, 150. mu.l of dimethyl sulfoxide (DMSO) was added to each well to dissolve the precipitate sufficiently, and the absorbance value (A) was measured at 490 nm. The results are shown in Table 1.

TABLE 1 Absorbance values for each group

As can be seen from the results in table 1, HSF cells cultured with exosomes were able to significantly stimulate proliferation of the cells, whereas exosomes obtained by saponin culture had better activity than exosomes cultured without saponin. In addition, the polypeptide has a better effect of stimulating HSF proliferation than an exosome, and after the polypeptide is introduced into the exosome, the polypeptide has an obvious effect of synergistically promoting HSF cell proliferation, and the absorbance value of the polypeptide reaches 1.45 +/-0.12, which is obviously improved compared with a positive control.

Example 6 mouse experiments

150 BALB/c mice were randomly divided into 5 groups, i.e., a control group; a model group; ③ a group of polypeptides; EXAMPLE 3 exosome treatment group; example 4 exosome treatment group; each group had 30. Preparing a chronic skin ulcer mouse model by adopting a method of immunosuppression composite skin wound for each group except a control group, and carrying out intramuscular injection of hydrocortisone for 0.5mg/d for 7 days before the experiment; the blank mice were injected intramuscularly with 0.2ml of physiological saline. In the experiment, pentobarbital sodium 60mg/kg is given for abdominal anesthesia, the back is unhaired and disinfected, a round hole is punched on the back of a mouse by a special puncher, and the skin with the diameter of 11mm is cut and deeply buried under the skin. And (4) carrying out single-cage breeding after molding, and carrying out aseptic feed feeding in an SPF-level animal room. The wound of the treatment group is dripped with 50 mul of 5 multiplied by 10⁶The exosome of (1) or 50 μ l containing 100ug of polypeptide, wrapped with a disposable sterile dressing, changed the dressing 1 time for 3 days and photographed. After skin injury, the mice in the control group and the model group were injected with 0.25mg/d of physiological saline every other day intramuscularly, and the mice in the other groups were injected with the same dose of hydrocortisone 0.25mg/d every other day intramuscularly. Wound tissue was harvested at day 10 after sacrifice of the mouse skin wound.

The detection indexes and the method are (1) wound area is photographed 10 days after the mouse is modeled, and the wound area is calculated by using image analysis software IPP 5.1. The results are shown in FIG. 2.

As can be seen from FIG. 2, the difference between the wound surface areas of the control group and the model group was statistically significant at 10 days (P < 0.01). Compared with the model group, the wound surface areas of the exosome group and the polypeptide group are obviously reduced, which shows that the polypeptide and the exosome can obviously reduce the wound surface area, particularly the exosome introduced with the polypeptideThe wound area is only 12.0 +/-1.0 mm²And has smaller wound area.

(2) TNF-alpha (radioimmunity method) is prepared by taking granulation tissue of wound, weighing, homogenizing in 1ml physiological saline, centrifuging at 3000r/min for 20min, collecting supernatant, operating according to radioimmunity specification, and measuring radioactivity count with gamma-counter. The results are shown in FIG. 3.

The TNF-alpha level of the model group is higher than that of the control group 10 days after the trauma, the TNF-alpha level of each group of the polypeptide group and the exosome group is obviously reduced compared with that of the model group (P is less than 0.01), and particularly, the TNF-alpha level of the exosome group introduced with the polypeptide is only (9.1 +/-0.9) ng/mL, as shown in figure 3.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; the reference to reagent concentrations and other parameters in the examples is one possible way under the inventive concept and is not limited to the relevant experimental parameters defined in the examples; under the conception of the invention, the related technical problems can be solved, and the related experimental parameters for achieving the technical effects of the invention are all within the protection scope of the invention; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Beijing Yunshenda Biotechnology development Co., Ltd

<160> 1

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Trp Tyr Gln His Ile Gly Val Gln Pro Met Tyr Trp Pro Ser Gly Tyr

1 5 10 15

Claims

1. An application of an exosome introduced with active peptide in preparing cosmetics for repairing skin injury, promoting fibroblast proliferation and preventing wrinkle generation is characterized in that: the sequence of the active peptide is shown as SEQ ID NO: 1, wherein the exosome is a placental stem cell exosome.

2. Use according to claim 1, characterized in that: wherein the preparation method of the exosome introduced with the active peptide comprises the following steps: exosomes were dissolved in 50mM trehalose in PBS buffer; converting SEQ ID NO: 1, adding the active peptide into the buffer solution, uniformly mixing, and transferring into an electric rotating cup for electric rotation, wherein the electric rotating cup is used for electric rotation under the conditions of 400V voltage, 175 muF capacitance and 41Tim capacitance; then filtering with an inverted centrifugal ultrafiltration membrane to remove free and non-specifically bound polypeptide, thus obtaining the exosome into which the polypeptide is introduced; wherein the exosome is a placental stem cell exosome, and the preparation method of the exosome comprises the following steps: taking placental stem cells, replacing an exosome secretion promoting culture medium when the cells are fused and grown to 85 percent: culturing 15% AGS, 0.5% total ginsenoside with 84.5% DMEM/F12 for 60h, and collecting cell supernatant; centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 10min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, filtering with 0.22 μm needle filter to remove microbubbles and possible apoptotic bodies, transferring the supernatant to an ultracentrifuge tube with 20mL empty needle, and centrifuging at 10 mL empty needle⁶Centrifuging for 70min at x g, removing supernatant, collecting precipitate to obtain crude extract exosome, dissolving with PBS, mixing, and further 10 times⁶Xg centrifugation for 70min, supernatant removed, 100. mu.L PBS dissolved precipitation, obtained relatively pure exosomes.

3. Use according to claim 2, characterized in that the cosmetic product is a cream or lotion.

4. Use according to claim 2, characterized in that the cosmetic product comprises a gelling polymer.

5. The use according to claim 4, wherein the gelling polymer is at least one of agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum and locust bean gum.

6. An application of an exosome introduced with active peptide in preparing a pharmaceutical composition for repairing skin injury, promoting fibroblast proliferation and preventing wrinkle generation, which is characterized in that: the sequence of the active peptide is shown as SEQ ID NO: 1, wherein the exosome is a placental stem cell exosome.

7. Use according to claim 6, characterized in that: wherein the preparation method of the exosome introduced with the active peptide comprises the following steps: exosomes were dissolved in 50mM trehalose in PBS buffer; converting SEQ ID NO: 1, adding the active peptide into the buffer solution, uniformly mixing, and transferring into an electric rotating cup for electric rotation, wherein the electric rotating cup is used for electric rotation under the conditions of 400V voltage, 175 muF capacitance and 41Tim capacitance; then filtering with an inverted centrifugal ultrafiltration membrane to remove free and non-specifically bound polypeptide, thus obtaining the exosome into which the polypeptide is introduced; wherein the exosome is a placental stem cell exosome, and the preparation method of the exosome comprises the following steps: taking placental stem cells, replacing an exosome secretion promoting culture medium when the cells are fused and grown to 85 percent: culturing 15% AGS, 0.5% total ginsenoside with 84.5% DMEM/F12 for 60h, and collecting cell supernatant; centrifuging at 300 Xg for 10min to remove dead cells and large cell debris, centrifuging at 2000 Xg for 10min to remove dead cells and cell debris, centrifuging at 10000 Xg/min for 30min to remove vesicles with large cell debris, filtering with 0.22 μm needle filter to remove microbubbles and possible apoptotic bodies, transferring the supernatant to an ultracentrifuge tube with 20mL empty needle, and centrifuging at 10 mL empty needle⁶Centrifuging for 70min at x g, removing supernatant, collecting precipitate to obtain crude extract exosome, dissolving with PBS, mixing, and addingBy 10⁶Xg centrifugation for 70min, supernatant removed, 100. mu.L PBS dissolved precipitation, obtained relatively pure exosomes.

8. The use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.