CN114106205A

CN114106205A - Preparation of mesenchymal stem cell exosome and composition and application of composition in cosmetics

Info

Publication number: CN114106205A
Application number: CN202111445023.XA
Authority: CN
Inventors: 付强
Original assignee: Shandong Sileji Pharmaceutical Technology Co ltd; Binzhou Medical College
Current assignee: Shandong Sileji Pharmaceutical Technology Co ltd; Binzhou Medical College
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-03-01
Anticipated expiration: 2041-11-30
Also published as: CN114106205B

Abstract

The invention discloses a mesenchymal stem cell exosome and a preparation method of a composition and application of the composition in cosmetics, the invention modifies mesenchymal stem cells through TRADD and cultures the mesenchymal stem cells, then extracts exosome from adipose mesenchymal stem cells by utilizing a microfluidic technology, and combines the exosome with coupling polypeptide of palmitoyl-7 peptide and TH peptide to prepare novel cosmetics which can be used for skin aging resistance, wrinkle resistance, oxidation resistance and repair; compared with the prior art, the preparation method has the advantages that the purification rate of exosomes from mesenchymal stem cells is higher, the targeting property and the penetrability are stronger, the skin thickness can be increased by coupling polypeptide, the wrinkle depth is reduced, the epidermis is compact, the absorption effect of plant components can be enhanced by combining the coupled polypeptide and the skin thickness, and the preparation method is innovative.

Description

Preparation of mesenchymal stem cell exosome and composition and application of composition in cosmetics

Technical Field

The invention relates to the technical field of cosmetics, in particular to a preparation method of a mesenchymal stem cell exosome and a composition and application of the composition in cosmetics.

Background

Palmitoyl tetrapeptide-7, also called palmitoyl tetrapeptide-3, has an amino acid sequence of Pal-Gly-Gln-Pro-Arg, abbreviated as Pal-GQPR, belongs to one of the series of palmitoyl oligopeptide polypeptides in signal peptides, and palmitoyl tetrapeptide-7 can accelerate the expression of granulocyte chemotactic protein (GCP-2) and promote wound recovery, and through the research on the peptide, palmitoyl tetrapeptide-7 can stimulate the regeneration of collagen fibers in dermis by acting as a cell messenger. Palmitoyl tetrapeptide 7 is also believed to increase the hyaluronic acid content in the skin, and thus may help tighten the skin by drawing moisture to the epidermis. In addition, palmitoyl tetrapeptide-7 is a fragment of immunoglobulin IgG, which has many bioactive functions, particularly immunomodulatory functions.

The cell-penetrating peptide is a cationic short peptide consisting of 10-30 amino acid residues, can mediate various substances to enter cells, such as DNA, protein, antibody, imaging agent, nanoparticle, liposome and the like, TH is used as one of the cell-penetrating peptides, TK [ AGYLLGKINLKKLAKL(Aib) KKIL-NH2] is used as a template, all lysine in the sequence is replaced by histidine, and a novel cell-penetrating peptide with pH selectivity and low toxicity is synthesized.

The palmitoyl tetrapeptide-7 and the TH peptide are coupled to form the polypeptide coupled polypeptide, a new method for modifying the palmitoyl tetrapeptide is explored, the property of the palmitoyl tetrapeptide is improved, the targeting property is improved, the stability and the safety of the palmitoyl tetrapeptide are improved while the activity is kept, the toxicity to normal cells is reduced while the self effect is exerted efficiently.

Exosomes are small vesicles of uniform size, about 30-200nm in diameter, of lipid bilayer structure, actively secreted out of cells by most types of cells, and widely present in many biological fluids. As a carrier for intercellular communication information exchange, exosomes have practical values like plasma, lymph fluid and the like, but the extraction and separation method and efficiency thereof are important problems influencing the application. Due to the size of exosome in nanometer level, the efficient and rapid extraction and separation method has been a problem which is not completely solved. Currently, the commonly used methods for exosome extraction are: ultracentrifugation precipitation, ultrafiltration, commercial kit methods, novel immunoaffinity capture methods, microfluidic technologies, and the like. The methods have the advantages and the disadvantages, the method has wide range of exosome extraction by using the microfluidic technology in recent years, the microfluidic technology can separate high-purity exosomes from biological fluids with high yield, the technology is easy to use, has high flux and can be automated, and the technology can be used as a new direction for researching the exosome extraction.

The skin is the largest organ of the human body, located on the outermost surface of the human body structure. The main functions of the medicine are to play a role of immune barrier, protect human body from invasion of external harmful substances, pathogens and the like, and also have the functions of regulating the metabolism of the organism, feeling touch and the like. However, skin is often damaged by various intrinsic pathologies and external mechanical factors, these acute and chronic skin defects place a heavy burden on patients and society, and skin inflammation and skin barrier breakdown are characteristic of most skin problems. Anti-inflammation and repair of the skin barrier have become central to current skin care. In addition, the skin has the problem of scars, once the scars are generated, the scars cannot be completely eliminated, so that the formation of the scars is particularly important to prevent, the proliferation of fibroblasts and the synthesis of collagen are suppressed from the source only by searching for the gene defects of the scars and correcting the gene defects from the gene level, the generation and the development of the scars are avoided or reduced, the effects of exosomes secreted by different stem cells possibly have different effects, and the cosmetics for synergistically resisting aging, allergy and repairing are not enough at present. Therefore, it is an object of the art to provide a new exosome composition cosmetic.

Disclosure of Invention

The invention aims to provide a preparation method of a mesenchymal stem cell exosome and a composition coupling polypeptide, and provides application of the composition in the cosmetic fields of wrinkle resistance, allergy resistance, healing promotion and the like.

In order to achieve the purpose, the invention provides the following technical scheme: a mesenchymal stem cell exosome and a composition thereof comprise a coupling polypeptide, wherein the molecular structural formula of the coupling polypeptide is as follows:

preferably, the preparation method of the conjugated polypeptide comprises the following steps: (1) synthesizing a TH peptide using a polypeptide solid phase synthesis method; (2) dissolving TH peptide and palmitoyl tetrapeptide-7 in a methanol solvent according to a molar ratio of 1:1.2, and coupling under the protection of a catalyst and inert gas to enable an N-terminal amino group of the TH peptide and a carboxyl terminal of palmitoyl tetrapeptide-7 to form an amido bond for connection, thereby preparing the TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide.

Preferably, the preparation method of the mesenchymal stem cell exosome comprises the following steps: (1) TRADD modified mesenchymal stem cell: the TRADD overexpression adenovirus is constructed, the obtained TRADD overexpression adenovirus is used for transfecting mesenchymal stem cells, and experimental research proves that the TRADD gene can inhibit the formation of skin scars and improve the flatness and color of the skin scars, so the TRADD gene is selected to construct the adenovirus; (2) culturing mesenchymal stem cells: culturing 3-5 generations of TRADD over-expressed mesenchymal stem cells by using a low-sugar DMEM culture medium containing fetal calf serum with the volume concentration of 10%, changing a serum-free culture medium for culturing for 48h when the cells are fused to 70% -80%, and collecting the cultured supernatant; (3) separating the mesenchymal stem cell exosomes by a microfluidic technology, and purifying to obtain the TRADD modified mesenchymal stem cell exosomes; the mesenchymal stem cells are adipose mesenchymal stem cells.

Preferably, the micro-fluidic chip is mainly used for separating the mesenchymal stem cell exosome and comprises a PDMS (polydimethylsiloxane) substrate and a PC (polycarbonate) membrane, a bottom cover is arranged on the membrane and is made of a PC material, the substrate, the membrane and the bottom cover are connected through adhesive tape sealing and laminating, a sample inlet, a bent channel, a micro channel and a liquid outlet are arranged on the membrane, wherein a first sample inlet and a second sample inlet are respectively connected with a second micro channel and a third micro channel through the bent channel, the second micro channel and the third micro channel are horizontally arranged in parallel, the other end of the second micro channel is connected with a waste groove through the liquid outlet, the other end of the third micro channel is connected with a recovery groove through the liquid outlet, a first micro channel is further arranged at the vertical position of the second micro channel and the third micro channel, a positive electrophoresis groove and a negative electrophoresis groove are respectively arranged at the two ends of the first micro channel, and a nano-pore film is further arranged at the joint of the third micro-channel and the first micro-channel, and the lower end of the nano-pore film is an enrichment pool.

Preferably, the first sample inlet and the second sample inlet can be connected with a micro-injection pump, the flow rate of the pump is 0.1mL/h, and the pore diameter of the first sample inlet and the second sample inlet is 0.8 μm.

Preferably, the length of the first microchannel is 55mm, the width of the first microchannel is 5mm, and the depth of the first microchannel is 100 μm, the length of the second microchannel and the length of the third microchannel are 48mm, the width of the second microchannel and the width of the third microchannel are 2mm, and the depth of the third microchannel is 100 μm, the size of the nanopore membrane is 5mm x 2mm, and the pore diameter of the nanopore membrane is 30-200 nm.

Preferably, the diameter of the prepared mesenchymal stem cell exosome is within the range of 55-100 nm.

Preferably, the cosmetics containing the mesenchymal stem cell exosomes and the composition comprise the following components: the composition comprises mesenchymal stem cell exosomes, TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide, olive fruit powder, red grape powder, a tree peony bark extract, an emulsifier, a humectant, a thickener, a chelating agent and the balance of a solvent.

Preferably, the cosmetic comprises one or more of the following uses: (1) the application in cosmetics for resisting skin aging, wrinkle, oxidation and repairing regeneration; (2) the application in cosmetics for promoting skin scar and wound healing; (3) use in antiallergic and antiinflammatory cosmetic is provided.

Preferably, the formulation of the cosmetic may be selected from any of the following forms: A. using mesenchymal stem cell exosomes as additives: an additive with the mesenchymal stem cell exosome as an effective component; b: a freeze-dried powder preparation: and (4) carrying out freeze-drying dormancy and freeze-drying on the exosome concentrated solution to prepare a dry powder preparation.

Compared with the prior art, the invention has the beneficial effects that:

1. compared with the prior art, the invention adopts palmitoyl-7 peptide as a main carrier material and adopts membrane-penetrating active peptide TH for coupling, so that on one hand, the targeting property and the penetrating capability are strong, the structure is more stable, on the other hand, the N-terminal amino group of the TH peptide is connected with the carboxyl group of the palmitoyl-7 peptide through amido bond, the activity of the palmitoyl-7 peptide is reserved, and the modification is carried out through a solid-phase synthesis method, so that the biological safety of cells is improved.

2. According to the invention, the TRADD gene is overexpressed in ADSC cells, and then the ADSC-derived exosomes are obtained through separation and extraction, the TRADD gene overexpression lentivirus can improve the flatness and color uniformity of skin scars, and the mesenchymal stem cells are modified to highly express TRADD and then used as cosmetic raw materials, so that the improvement effect of exosomes on human skin and scars can be obviously improved.

3. The separation of the exosomes in the invention innovatively uses a microfluidic technology, and the separation of the exosomes can be rapidly and efficiently realized by adopting a portable and small chip.

4. The invention adds the ADSC-derived exosome and the coupling polypeptide in the application of the cosmetics, can effectively resist aging, improve skin scars and wrinkles, and improve anti-allergic reaction, and the ADSC-derived exosome can also promote the absorption of plant extracts and improve the utilization rate of plant components in the skin.

Other features of the present disclosure and advantages thereof will become apparent from the following detailed description of exemplary embodiments thereof, which proceeds with reference to the accompanying drawings.

Drawings

In order to more clearly illustrate the embodiments of the present disclosure or technical solutions in related arts, the drawings used in the description of the embodiments or related arts will be briefly introduced below, it is obvious that the drawings in the following description are only embodiments of the present disclosure, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of a chip used in the microfluidic technology of the present invention;

FIG. 2 is a graph of the mean diameter of the exosomes obtained for the present invention;

in the figure: 1. the electrophoresis device comprises a positive electrode electrophoresis tank, 2, a negative electrode electrophoresis tank, 3, a first micro-channel, 4, a first sample inlet, 5, a second micro-channel, 6, a bent channel, 7, a second sample inlet, 8, a third micro-channel, 9, a recovery tank, 10, an enrichment pool, 11, a waste tank, 12 and a nanopore film.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise. Meanwhile, it should be understood that the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description. Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate. In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, further discussion thereof is not required in subsequent figures.

Spatially relative terms, such as "above … …," "above … …," "above … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial relationship to another device or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is turned over, devices described as "above" or "on" other devices or configurations would then be oriented "below" or "under" the other devices or configurations. Thus, the exemplary term "above … …" can include both an orientation of "above … …" and "below … …". The device may be otherwise variously oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

A mesenchymal stem cell exosome and a composition thereof comprise a coupling polypeptide, wherein the molecular structural formula of the coupling polypeptide is as follows:

the invention provides a preparation method of the conjugated polypeptide, which comprises the following steps: (1) synthesizing a TH peptide using a polypeptide solid phase synthesis method; (2) dissolving TH peptide and palmitoyl tetrapeptide-7 in a methanol solvent according to a molar ratio of 1:1.2, and coupling under the protection of a catalyst and inert gas to enable an N-terminal amino group of the TH peptide and a carboxyl terminal of palmitoyl tetrapeptide-7 to form an amido bond for connection, thereby preparing the TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide.

The peptide in the step (1) is a cell-penetrating active peptide TH, a novel cell-penetrating peptide with pH selectivity and low toxicity, and the specific sequence is AGYLLGHINLHHLAHL(Aib) HHIL-NH₂。

The coupling reaction of the invention is realized by the following conditions: adding TH peptide, palmitoyl tetrapeptide-7 and N, N-Diisopropylhexylamine (DIEA) in a molar ratio of 1:2:3 into a 50mL reaction bottle, adding 20% of N, N-dimethylformamide for dissolving, reacting for 8-12 hours at 25 ℃ under the protection of nitrogen, determining the molecular weight by using MS-IT-TOF, and purifying the crude product by using HPLC to obtain the coupled polypeptide of the TH peptide and the palmitoyl tetrapeptide-7.

Another aspect of the present invention provides a method for preparing a mesenchymal stem cell exosome, comprising:

(1) TRADD modified mesenchymal stem cell: constructing TRADD over-expression adenovirus, and transfecting mesenchymal stem cells by using the obtained TRADD over-expression adenovirus; the mesenchymal stem cell is an adipose mesenchymal stem cell, and the adipose mesenchymal stem cell is derived from a human body; the modification method of the present invention is not particularly limited, and any method known in the art may be used.

(4) Culturing mesenchymal stem cells: culturing human ADSC in a serum-free culture system by adopting a feeder-free culture method, culturing 3-5 generations of TRADD over-expressed mesenchymal stem cells by using a low-sugar DMEM culture medium containing fetal calf serum with the volume concentration of 10%, culturing for 48h by using the serum-free culture medium when the cells are fused to 70% -80%, centrifuging for 20min by 2000g of centrifugal force, filtering by using a 0.22 micron filter membrane to remove the cells, collecting the cultured supernatant, and purifying exosomes in the culture medium, namely adipose mesenchymal stem cell exosomes; the serum-free culture system is selected from commercial culture medium with model number TeSRTM-E8TM or mTeSR 1.

(5) Separating the mesenchymal stem cell exosomes by a microfluidic technology, and purifying to obtain the TRADD modified mesenchymal stem cell exosomes;

concretely, the micro-fluidic chip is mainly adopted to the mesenchymal stem cell exosome by the micro-fluidic technology, referring to fig. 1, the micro-fluidic chip is composed of a PDMS (polydimethylsiloxane) substrate and a PC (polycarbonate) membrane, the membrane is provided with a bottom cover, the bottom cover is also made of PC material, the substrate, the membrane and the bottom cover are connected by adhesive tape sealing and laminating, the membrane is provided with a sample inlet, a bent channel, a micro-channel and a liquid outlet, wherein a first sample inlet 4 and a second sample inlet 7 are respectively connected with a second micro-channel 5 and a third micro-channel 8 through a bent channel 6, the second micro-channel 5 and the third micro-channel 8 are arranged in parallel with each other horizontally, the other end of the second micro-channel 5 is connected with a waste groove 11 through the liquid outlet, the other end of the third micro-channel 8 is connected with a recycling groove 9 through the liquid outlet, the perpendicular position of the second micro-channel 5 and the third micro-channel 8 is also provided with a first micro-channel 3, the two ends of the first microchannel 3 are respectively provided with a positive electrode electrophoresis tank 1 and a negative electrode electrophoresis tank 2, the joint of the third microchannel 8 and the first microchannel 3 is also provided with a nanopore film 12, and the lower end of the nanopore film 12 is provided with an enrichment pool 10.

Further, the first sample inlet 4 and the second sample inlet 7 can be connected with a micro-injection pump, the flow rate of the pump is 0.1mL/h, and the aperture of the first sample inlet 4 and the aperture of the second sample inlet 7 are 0.8 μm.

Furthermore, the length of the first microchannel 3 is 55mm, the width thereof is 5mm, and the depth thereof is 100 μm, the length of the second microchannel 5 and the third microchannel 8 is 48mm, the width thereof is 2mm, and the depth thereof is 100 μm, the size of the nanopore membrane 12 is 5mm × 2mm, and the pore diameter of the nanopore membrane 12 is 30-200 nm.

The working principle is as follows: the chip is designed to extract exosomes by utilizing that the exosomes have negative charges on the surface and can move directionally under the action of an electric field, when a mixture containing the exosomes enters the chip, the exosomes with the negative charges can move directionally under the action of the electric field and enter a channel filled with agarose, and as the internal pore diameter of the agarose is 200-300nm, a target exosome can pass through the agarose gel, and extracellular vesicles with larger sizes cannot pass through the agarose gel and enter a waste groove 11 from an outlet of a second microchannel 5. Under the action of an electric field, exosomes with the size of 30-200nm are driven to the nano-pore membrane 12 to be captured, free biological small molecules and other small vesicles penetrate through the nano-pore membrane 12 and enter the enrichment pool 10, and after electrophoresis is finished, the exosomes can be taken out from the recovery tank 9, so that the separation and purification of the exosomes are realized.

The purification method comprises the following steps: adding equal volume of a first sodium chloride solution (16% (w/v) polyethylene glycol 6000 and 1M sodium chloride) containing polyethylene glycol 6000 into the supernate containing the exosomes, standing for more than 12 hours, centrifuging 3500g for 30min, collecting the precipitate, suspending the precipitate in PBS buffer solution, adding equal volume of a second sodium chloride solution (10% (w/v) polyethylene glycol 6000 and 1M sodium chloride) containing polyethylene glycol 6000 into the supernate, standing for more than 4 hours, centrifuging 3500g for 30min, collecting the precipitate, suspending the precipitate in PBS buffer solution, and obtaining the purified exosomes.

The diameter of the obtained adipose-derived mesenchymal stem cell exosome is measured, the measuring method is not particularly limited, and the method known in the field, such as a transmission electron microscope or a nanoparticle analysis system, is adopted, so that the diameter of the adipose-derived mesenchymal stem cell exosome prepared by the method is measured to be within the range of 55-100nm, and the average diameter is 63.65 nm.

The invention also provides an application of the adipose-derived mesenchymal stem cell exosome and the conjugated polypeptide as a composition in cosmetics, which mainly comprises the following components: the composition comprises mesenchymal stem cell exosomes, TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide, olive fruit powder, red grape powder, a tree peony bark extract, an emulsifier, a humectant, a thickener, a chelating agent and the balance of a solvent.

In the mixture ratio, the weight ratio of the exosome to the coupling polypeptide to the plant components is 1: 2-3: 3, the weight ratio of the exosome to the coupling polypeptide is 1:3, and the weight ratio of olive fruit: red grape powder: the weight ratio of the moutan bark extract is 1:2: 2.

The invention also details the use of the cosmetic, including one or more of the following aspects: (1) the application in cosmetics for resisting skin aging, wrinkle, oxidation and repairing regeneration; (2) the application in cosmetics for promoting skin scar and wound healing; (3) use in antiallergic and antiinflammatory cosmetic is provided.

In addition, the preparation of the cosmetic can be selected from any one of the following forms: A. using mesenchymal stem cell exosomes as additives: an additive with the mesenchymal stem cell exosome as an effective component; b: a freeze-dried powder preparation: and (4) carrying out freeze-drying dormancy and freeze-drying on the exosome concentrated solution to prepare a dry powder preparation. In the embodiment of the invention, the additive containing the ADSC exosomes can be used for preparing various cosmetics, such as facial masks, face creams, milky lotions, essence, eye creams, stock solutions and the like.

Example 1:

referring to fig. 1, a specific operation method of the microfluidic chip: (1) opening the bottom cover of the chip, sticking completely matched common adhesive tapes at the windows of the two horizontal channels, and sealing by using the bottom cover; (2) accurately weighing 0.1g of agarose gel into a conical flask, adding 10mL of 1 xTAE buffer solution, placing the conical flask in a microwave oven for heating and dissolving, preparing 1.0% agarose gel, injecting the agarose gel into a chip from the positive electrode of an electrophoresis tank to completely fill a first microchannel of the chip, placing the chip at 4 ℃ for about 20min, opening a bottom cover after the gel is completely solidified, taking out a common adhesive tape and the gel to form two channels, namely a second microchannel and a third microchannel, wherein the second microchannel is connected with a micro-injection pump, and the third microchannel is used for enriching exosomes; (3) injecting PBS eluent with pH value of 6.0 into the chip from the second sample inlet and allowing the chip to enter an enrichment pool below the nanopore film; (4) loading about 80 mu L of human ADSC into an injector, connecting the injector to a micro-injection pump, filling 1 XTAE buffer solution into the anode and the cathode of an electrophoresis tank, adjusting the voltage of the electrophoresis tank to be 100-150V, and opening the injection pump while applying voltage to make the exosome in the human ADSC move directionally under the action of an electric field, pass through agarose gel and be captured by a nanopore membrane, thereby completing the extraction of the exosome. And the diameter of the extracted exosomes was measured, and the result showed that the particle size ranged from 55 to 100nm, preferably 55 to 65nm, see fig. 2.

Example 2:

1-3 parts of mesenchymal stem cell exosome, 2-9 parts of TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide, 1-3 parts of olive powder, 2-6 parts of red grape powder, 2-6 parts of moutan bark extract, 0-1 part of glyceryl stearate, 0-1 part of cetyl stearyl alcohol, 1-2 parts of urea, 0-1 part of glycerol, 1-1.5 parts of isopropyl ester, 0-0.05 part of carbomer, 0-0.5 part of xanthan gum and the balance of deionized water are used for preparing the face cream, and the preparation method is a conventional method.

The specific operation method comprises melting purified exosome concentrated solution or exosome concentrated solution frozen at-80 deg.C in water bath, adding lyophilized excipient for dissolving, filtering, and lyophilizing in a lyophilizer with lyophilization vacuum degree of 0.04mbar and lyophilization time of 10-20 hr.

Example 3: anti-aging test Using the cream of example 2

Human skin aging is a complex process influenced by many factors, and at present, the aging damage of the skin is mainly caused by accumulation of free radicals, and SOD is the only metalloenzyme taking the Inula nervosa free radicals as a substrate in vivo and can effectively remove the free radicals, so the SOD level is taken as an anti-aging index.

60 female mice were selected and randomly divided into 3 groups: normal control group, invention group and market product group, wherein the market product is the same kind of anti-aging product from regular market, normal control group is fed with normal saline, the other groups of animals are fed with the invention product and market product, after last administration for 1h, blood is taken from eye orbit of mouse, centrifugation is carried out, and supernatant is taken to measure SOD value.

TABLE 1 anti-aging results for cosmetics prepared according to the invention

Group of	SOD value (U/mg)
		Normal control group	138.85±12.44
Group of the invention	159.63±15.36^**
		Group of products on market	140.25±13.57^*

As can be seen from the above table, compared with the control group, the SOD content of the products of the invention group and the market group is obviously increased (P < 0.01), while the difference between the products of the invention group and the market group is obvious (P <0.05), and the SOD value of the product of the invention group is obviously higher than that of the market group, which shows that the products of the invention group can obviously increase the SOD content.

Example 4: scar test Using the cream of example 2

By adopting the skin scar test method in the invention patent CN 110123838A, female C57BL/6 mice of 7-8 weeks old are randomly divided into a control group, a skin scar model group and a treatment group, 10 mice are selected for each group, and the dermis thickness measurement is carried out on the mice, the result shows that the dermis thickness of the skin scar model group is (430.5 +/-36.4 mu m) and is obviously higher than that of the treatment group (287.3 +/-10.2 mu m, P is less than 0.05), so the treatment group can prevent the formation of scars.

Example 5: anti-allergy test Using the cream of example 2

The mice of each half of the male and female are randomly divided into four groups, each group comprises 12 mice, the first group is a normal control group, the second group is a positive control group, 1% DNCB is applied for induction, 1% OX is adopted for enhanced induction, repeated induction is carried out for sensitization, the third group and the fourth group are treatment groups, the third group is treated by using compound phellodendron bark lotion, the fourth group is treated by using the face cream of the invention and is smeared three times every day, the skin of the mice of the normal control group is smooth, has no erythema, exudation and desquamation, the mice of the positive control group have the phenomena of erythema, pimple, exudation and the like, the skin tissue state of the mice of the treatment groups is obviously improved, the experimental effect of the invention group is not much different from the treatment effect of the medicine, the mice of the four groups are respectively subjected to serum total IGE level test, the result is shown in Table 2, and the IGE level of the positive control group is obviously improved compared with the normal control group, the level of IGE in the treated group was reduced compared to the positive control group, and there was no statistical difference between the level of IGE in the creams of the invention and the drug treatment.

Table 2 shows the results of the anti-sensitivity test of the cosmetics prepared according to the present invention

Claims

1. Mesenchymal stem cell exosomes and compositions, including mesenchymal stem cell exosomes and conjugated polypeptides, characterized in that: the molecular structural formula of the conjugated polypeptide is as follows:

2. a method of producing the conjugated polypeptide of claim 1, wherein: the preparation method of the conjugated polypeptide comprises the following steps:

(1) synthesizing a TH peptide using a polypeptide solid phase synthesis method;

(2) dissolving TH peptide and palmitoyl tetrapeptide-7 in a methanol solvent according to a molar ratio of 1:1.2, and coupling under the protection of a catalyst and inert gas to enable an N-terminal amino group of the TH peptide and a carboxyl terminal of palmitoyl tetrapeptide-7 to form an amido bond for connection, thereby preparing the TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide.

3. The method for preparing an exosome of mesenchymal stem cells according to claim 1, characterized by: the preparation method of the mesenchymal stem cell exosome comprises the following steps:

(1) TRADD modified mesenchymal stem cell: constructing TRADD over-expression adenovirus, and transfecting mesenchymal stem cells by using the obtained TRADD over-expression adenovirus;

(2) culturing mesenchymal stem cells: culturing 3-5 generations of TRADD over-expressed mesenchymal stem cells by using a low-sugar DMEM culture medium containing fetal calf serum with the volume concentration of 10%, changing a serum-free culture medium for culturing for 48h when the cells are fused to 70% -80%, and collecting the cultured supernatant;

(3) separating the mesenchymal stem cell exosomes by a microfluidic technology, and purifying to obtain the TRADD modified mesenchymal stem cell exosomes; the mesenchymal stem cells are adipose mesenchymal stem cells.

4. The method for preparing mesenchymal stem cell exosomes according to claim 3, wherein the method comprises the following steps: the mesenchymal stem cell exosome is separated by the microfluidic technology mainly adopts a microfluidic chip which is composed of a PDMS (polydimethylsiloxane) substrate and a PC (polycarbonate) membrane, a bottom cover is arranged on the membrane and is made of a PC material, the substrate, the membrane and the bottom cover are connected by adhesive tape sealing and laminating, a sample inlet, a bent channel, a micro channel and a liquid outlet are arranged on the membrane, wherein a first sample inlet (4) and a second sample inlet (7) are respectively connected with a second micro channel (5) and a third micro channel (8) through the bent channel (6), the second micro channel (5) and the third micro channel (8) are horizontally arranged in parallel, the other end of the second micro channel (5) is connected with a waste groove (11) through a liquid outlet, the other end of the third micro channel (8) is connected with a recovery groove (9) through a liquid outlet, second microchannel (5) and third microchannel (8) mutually perpendicular department still are equipped with first microchannel (3), the both ends punishment of first microchannel (3) are equipped with anodal electrophoresis tank (1) and negative pole electrophoresis tank (2) respectively, the junction of third microchannel (8) and first microchannel (3) still is equipped with nanopore film (12), the lower extreme of nanopore film (12) is for enriching pond (10).

5. The method for preparing mesenchymal stem cell exosomes according to claim 4, wherein the method comprises the following steps: the first sample inlet (4) and the second sample inlet (7) can be connected with a micro-injection pump, the flow rate of the pump is 0.1mL/h, and the aperture of the first sample inlet and the aperture of the second sample inlet are 0.8 mu m.

6. The method for preparing mesenchymal stem cell exosomes according to claim 4, wherein the method comprises the following steps: the length of the first micro-channel (3) is 55mm, the width is 5mm, the depth is 100 μm, the length of the second micro-channel (5) and the third micro-channel (8) is 48mm, the width is 2mm, the depth is 100 μm, the size of the nano-pore film (12) is 5mm x 2mm, and the pore diameter of the nano-pore film (12) is 30-200 nm.

7. The method for preparing mesenchymal stem cell exosomes according to claim 3, wherein the method comprises the following steps: the diameter of the prepared mesenchymal stem cell exosome is within the range of 55-100 nm.

8. The use of mesenchymal stem cell exosomes and compositions according to claim 1 in cosmetics, wherein: a cosmetic containing mesenchymal stem cell exosomes and a composition thereof comprises the following components: the composition comprises mesenchymal stem cell exosomes, TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide, olive fruit powder, red grape powder, a tree peony bark extract, an emulsifier, a humectant, a thickener, a chelating agent and the balance of a solvent.

9. Use of mesenchymal stem cell exosomes and compositions according to claim 8 in cosmetics, characterised in that: the application of the cosmetic comprises one or more of the following applications:

(1) the application in cosmetics for resisting skin aging, wrinkle, oxidation and repairing regeneration;

(2) the application in cosmetics for promoting skin scar and wound healing;

(3) use in antiallergic and antiinflammatory cosmetic is provided.

10. Use of mesenchymal stem cell exosomes and compositions according to claim 8 in cosmetics, characterised in that: the formulation of the cosmetic may be selected from any of the following forms:

A. using mesenchymal stem cell exosomes as additives: an additive with the mesenchymal stem cell exosome as an effective component;

B. a freeze-dried powder preparation: and (4) carrying out freeze-drying dormancy and freeze-drying on the exosome concentrated solution to prepare a dry powder preparation.