CN114106205B - Preparation of mesenchymal stem cell exosomes and compositions and application in cosmetics - Google Patents

Preparation of mesenchymal stem cell exosomes and compositions and application in cosmetics Download PDF

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CN114106205B
CN114106205B CN202111445023.XA CN202111445023A CN114106205B CN 114106205 B CN114106205 B CN 114106205B CN 202111445023 A CN202111445023 A CN 202111445023A CN 114106205 B CN114106205 B CN 114106205B
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mesenchymal stem
stem cell
microchannel
exosomes
cell exosomes
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CN114106205A (en
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付强
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Shandong Sileji Pharmaceutical Technology Co ltd
Binzhou Medical College
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Shandong Sileji Pharmaceutical Technology Co ltd
Binzhou Medical College
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    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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Abstract

The invention discloses a preparation method of mesenchymal stem cell exosomes and compositions and application thereof in cosmetics, wherein the mesenchymal stem cells are modified by TRADD, cultured and then extracted from adipose mesenchymal stem cells by utilizing a microfluidic technology, and the exosomes are combined with palmitoyl-7 peptide and TH peptide coupled polypeptides to prepare novel cosmetics which can be used for skin anti-aging, anti-wrinkle, anti-oxidation and repair; compared with the prior art, the invention has higher purification rate of exosomes derived from the intermediate stem cells, stronger targeting property and penetrability, and the coupled polypeptide can increase the skin thickness, reduce the wrinkle depth, compact epidermis and the like, and the combination of the two can strengthen the absorption effect of plant components, thereby having innovativeness.

Description

Preparation of mesenchymal stem cell exosomes and compositions and application in cosmetics
Technical Field
The invention relates to the technical field of cosmetics, in particular to preparation of mesenchymal stem cell exosomes and a composition and application thereof in cosmetics.
Background
Palmitoyl tetrapeptide-7, also known as palmitoyl tetrapeptide-3, has an amino acid sequence Pal-Gly-gin-Pro-Arg, abbreviated Pal-GQPR, belongs to one of the palmitoyl oligopeptide series of polypeptides in the signal peptide, and palmitoyl tetrapeptide-7 can accelerate the expression of granulocyte chemotactic protein (GCP-2) and promote wound healing, and by studying this peptide, it has been determined that palmitoyl tetrapeptide-7 can stimulate regeneration of collagen fibers in the dermis by acting as a cellular messenger. Palmitoyl tetrapeptide 7 is also believed to increase the hyaluronic acid content in the skin, thereby helping to tighten the skin by drawing moisture into the epidermis. In addition, palmitoyl tetrapeptide-7 is a fragment of immunoglobulin IgG, which has many bioactive functions, particularly immune modulating 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 taken as one of the cell penetrating peptides, TK [ AGYLLGKINLKKLAKL (Aib) KKIL-NH2] is taken as a template, all lysine in the sequence is replaced by histidine, and the novel cell penetrating peptide with pH selectivity and low toxicity is synthesized.
Palmitoyl tetrapeptide-7 and TH peptide are coupled to form polypeptide coupled polypeptide, a new mode of palmitoyl tetrapeptide modification 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 maintained, and the toxicity to normal cells is reduced while the palmitoyl tetrapeptide-7 plays a role in high efficiency.
Exosomes are small vesicles of lipid bilayer structure of uniform size, about 30-200nm in diameter, which are actively secreted extracellularly by most types of cells, widely present in many biological fluids. As a carrier for communication information exchange among cells, the practical value of exosomes is like plasma, lymph fluid and the like, but the extraction and separation method and efficiency are important problems affecting the application. Because of the nano-scale size of exosomes, the efficient and rapid extraction and separation method is a difficult problem which has not been thoroughly solved. The exosome extraction methods commonly used at present are as follows: ultracentrifugation precipitation, ultrafiltration, commercial kit methods, novel immunoaffinity capture methods, microfluidic techniques, and the like. The method has the advantages and disadvantages that the method has wider extraction range of exosomes by utilizing a microfluidic technology in recent years, the microfluidic technology can separate the exosomes with high purity from biological fluid with high yield, the technology is easy to use, high in flux and capable of being automated, and can be used as a new direction for related research of exosome extraction.
The skin is the largest organ of the human body, and is located outermost in the body structure. The main function of the compound is to play a role of immune barrier, protect human bodies from invasion of external harmful substances, pathogens and the like, also has the functions of regulating metabolism, sensing touch, etc. However, skin is often damaged by various intrinsic pathologies and external mechanical factors, and these acute and chronic skin defects place a heavy burden on the patient and society, skin inflammation and skin barrier disruption being a feature of most skin problems. Anti-inflammatory and repair of the skin barrier has become a major issue in current skin care. In addition, the skin has the problem of scar, once the scar is generated, the scar cannot be completely eliminated, so that the prevention of the scar formation is particularly important, only by searching for the scar gene defect and correcting the scar gene level, the scar is inhibited from being generated into fibroblast proliferation and collagen synthesis from the source, the occurrence and development of the scar are avoided or reduced, the effect of the exosomes secreted by different stem cells possibly has different effects, and the cosmetics for synergizing anti-aging, anti-sensitization and repairing are not enough at present. It is therefore a goal of the art to provide a new exosome composition cosmetic.
Disclosure of Invention
The invention aims to provide a preparation method of mesenchymal stem cell exosomes and composition coupled polypeptides, and provides application of the composition in the fields of cosmetics such as anti-wrinkle, antiallergic and healing promoting.
In order to achieve the above purpose, the present invention provides the following technical solutions: a mesenchymal stem cell exosome and a composition thereof, comprising a conjugated polypeptide having the molecular structural formula:
preferably, the method for preparing the coupled 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 performing coupling under the protection of a catalyst and inert gas to ensure that the N-terminal amino group of the TH peptide and the carboxyl terminal of the palmitoyl tetrapeptide-7 form an amide bond to be connected, so as to prepare the coupling polypeptide of the TH peptide and the palmitoyl tetrapeptide-7.
Preferably, the preparation method of the mesenchymal stem cell exosome comprises the following steps: (1) TRADD modified mesenchymal Stem cells: constructing TRADD over-expression adenovirus, and transfecting mesenchymal stem cells by using the obtained TRADD over-expression adenovirus, wherein experimental researches prove that the TRADD gene can inhibit skin scar formation and improve the evenness and the color of the skin scar, so that the TRADD gene is selected to construct the adenovirus; (2) mesenchymal stem cell culture: culturing 3-5 generations of mesenchymal stem cells over-expressed by TRADD by using a low-sugar DMEM culture medium containing 10% fetal calf serum by volume concentration, culturing for 48 hours by changing a serum-free culture medium when the cells are fused to 70% -80%, and collecting supernatant after culturing; (3) Separating and purifying the mesenchymal stem cell exosomes by a microfluidic technology to obtain the TRADD modified mesenchymal stem cell exosomes; the mesenchymal stem cells are adipose mesenchymal stem cells.
Preferably, the microfluidic technology is used for separating mesenchymal stem cell exosomes, the microfluidic chip is mainly composed of a PDMS (polydimethylsiloxane) substrate and a PC (polycarbonate) membrane, a bottom cover is arranged on the membrane, the bottom cover is also made of PC material, the substrate, the membrane and the bottom cover are connected through sealing and attaching by adhesive tapes, a sample inlet, a bent channel, a micro-channel and a liquid outlet are arranged on the membrane, the first sample inlet and the second sample inlet are respectively connected with the second micro-channel and the 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 tank through the liquid outlet, the other end of the third micro-channel is connected with a recovery tank 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 tank and a negative electrophoresis tank are respectively arranged at the two ends of the first micro-channel, a hole film is further arranged at the joint of the third micro-channel and the first micro-channel, and the lower end of the nano-hole film is enriched.
Preferably, the first sample inlet and the second sample inlet can be connected with a microinjection 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.
Preferably, the length of the first micro-channel is 55mm, the width is 5mm, the depth is 100 μm, the length of the second micro-channel and the third micro-channel is 48mm, the width is 2mm, the depth is 100 μm, the size of the nano-pore film is 5mm x 2mm, and the pore diameter of the nano-pore film is 30-200nm.
Preferably, the diameter of the prepared mesenchymal stem cell exosome is in the range of 55-100 nm.
Preferably, a cosmetic comprising mesenchymal stem cell exosomes and a composition comprising the following components: mesenchymal stem cell exosomes, TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide, olive powder, red grape powder, tree peony bark extract, emulsifying agent, humectant, thickening agent, chelating agent and the balance of solvent.
Preferably, the cosmetic comprises one or several of the following uses: (1) The application of the anti-aging, anti-wrinkle, antioxidant and repairing regenerative cosmetics to the skin; (2) The application in cosmetics for promoting skin scar and wound healing; (3) application in antiallergic and anti-inflammatory cosmetics.
Preferably, the formulation of the cosmetic may be selected in any of the following forms: A. mesenchymal stem cell exosomes are used as additives: an additive comprising the mesenchymal stem cell exosomes as an active ingredient; b: lyophilized powder formulation: freeze-drying and dormancy and freeze-drying 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 the transmembrane active peptide TH for coupling, so that on one hand, the targeting property and the penetrating power are strong, the structure is more stable, and on the other hand, the N-terminal amino of the TH peptide is connected with the carboxyl of the palmitoyl-7 peptide through an amide bond, so that the activity of the palmitoyl-7 peptide is reserved, and the cell biosafety is improved by modifying through a solid-phase synthesis method.
2. The invention is characterized in that TRADD gene is over-expressed in ADSC cells, ADSC source exosomes are obtained by separation and extraction, the TRADD gene over-expressed slow virus can improve the flatness and color uniformity of skin scars, and the mesenchymal stem cells are modified to make them highly express TRADD and then used as cosmetic raw materials, thus the improvement effect of exosomes on human skin and scars can be remarkably improved.
3. According to the invention, a microfluidic technology is creatively used for separating the exosomes, and the exosomes can be rapidly and efficiently separated by adopting a portable small chip.
4. The ADSC-derived exosome and the coupling polypeptide are added in the application of cosmetics, so that the skin scar and wrinkle can be effectively prevented from aging, the antiallergic reaction can be improved, the absorption of the plant extract can be promoted by the ADSC-derived exosome, and the utilization rate of plant components in the skin can be improved.
Other features of the present disclosure and its advantages will become apparent from the following detailed description of exemplary embodiments of the disclosure, which proceeds with reference to the accompanying drawings.
Drawings
In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the related art, the drawings that are required to be used in the embodiments or the related technical descriptions will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present disclosure, and other drawings may be obtained from the provided drawings without inventive effort to those of ordinary skill in the art.
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 average diameter of exosomes obtained according to the invention;
in the figure: 1. the device comprises an anode electrophoresis tank, 2, a cathode 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 tank, 11, a waste tank, 12 and a nano-pore film.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise. Meanwhile, it should be understood that the sizes of the respective parts shown in the drawings are not drawn in actual scale for convenience of description. Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but should be considered part of the specification where appropriate. In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further discussion thereof is necessary in subsequent figures.
Spatially relative terms, such as "above … …," "above … …," "upper surface at … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial location relative 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 in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "above" or "over" other devices or structures would then be oriented "below" or "beneath" the other devices or structures. Thus, the exemplary term "above … …" may include both orientations of "above … …" and "below … …". The device may also be positioned in other different ways (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, comprising a conjugated polypeptide having the molecular structural formula:
the invention provides a preparation method of the coupled 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 performing coupling under the protection of a catalyst and inert gas to ensure that the N-terminal amino group of the TH peptide and the carboxyl terminal of the palmitoyl tetrapeptide-7 form an amide bond to be connected, so as to prepare the coupling polypeptide of the TH peptide and the palmitoyl tetrapeptide-7.
The peptide in the step (1) is a membrane penetrating active peptide TH, a novel cell membrane penetrating peptide with pH selectivity and low toxicity, and the specific sequence is AGYLLGHINLHHLAHL (Aib) HHIL-NH 2 。
The specific coupling reaction of the invention is realized by the following conditions: TH peptide, palmitoyl tetrapeptide-7 and N, N-Diisopropylhexylamine (DIEA) with a molar ratio of 1:2:3 are respectively added into a 50mL reaction bottle, 20% of N, N-dimethylformamide is added for dissolution, the mixture is reacted for 8 to 12 hours at 25 ℃ under the protection of nitrogen, the molecular weight is determined by using MS-IT-TOF, and the crude product is purified by using HPLC, so that the coupling polypeptide of the TH peptide and the palmitoyl tetrapeptide-7 is obtained.
Another aspect of the present invention provides a method for preparing mesenchymal stem cell exosomes:
(1) TRADD modified mesenchymal stem cells: constructing TRADD over-expression adenovirus, and transfecting mesenchymal stem cells by using the obtained TRADD over-expression adenovirus; the mesenchymal stem cells are adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells are derived from human bodies; the method of modification is not particularly limited, and methods known in the art may be employed.
(4) Mesenchymal stem cell culture: culturing human ADSC in a serum-free culture system by adopting a feeder layer-free culture method, culturing 3-5 generations of the culture medium with low-sugar DMEM containing 10% fetal bovine serum by volume concentration, constructing TRADD over-expressed mesenchymal stem cells, culturing for 48 hours by changing the serum-free culture medium when the cells are fused to 70% -80%, centrifuging for 20 minutes by 2000g centrifugal force, filtering and removing the cells by a 0.22-micrometer filter membrane, collecting the supernatant after the culture, and purifying exosomes in the culture medium, namely the adipose-derived mesenchymal stem cell exosomes; the serum-free culture system was selected from commercially available medium types TeSRTM-E8TM or mTESR 1.
(5) Separating and purifying the mesenchymal stem cell exosomes by a microfluidic technology to obtain the TRADD modified mesenchymal stem cell exosomes;
specifically, the microfluidic technology is used for separating mesenchymal stem cell exosomes, a microfluidic chip is mainly adopted, referring to fig. 1, the microfluidic chip is composed of a PDMS (polydimethylsiloxane) substrate and a PC (polycarbonate) membrane, a bottom cover is arranged on the membrane, the bottom cover is also made of a PC material, the substrate, the membrane and the bottom cover are in sealing fit connection through an adhesive tape, a sample inlet, a bent channel, a micro-channel and a liquid outlet are arranged on the membrane, 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 the liquid outlet, the other end of the third micro-channel 8 is connected with a recovery groove 9 through the liquid outlet, a first micro-channel 3 is further arranged at the vertical position of the second micro-channel 5 and the third micro-channel 8, a positive electrophoresis groove 1 and a negative electrophoresis groove 2 are respectively arranged at the two ends of the first micro-channel 3, the second micro-channel 8 and the third micro-channel 8 and the first micro-channel 8 are further provided with a nano-membrane 10 at the position of the nano-pore.
Further, the first sample inlet 4 and the second sample inlet 7 can be connected with a microinjection 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.
Further, 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 30-200nm.
Working principle: the use of the exosomes with negative charge on their surface, the directional movement of the chip under the action of an electric field is designed to extract exosomes, when the mixture containing exosomes enters the chip, the exosomes with negative charge can move directionally under the action of an electric field into the agarose filled channel, as the internal pore size of agarose is 200-300nm, the target exosomes can pass through the agarose gel, while the larger size extracellular vesicles cannot pass through the agarose gel and enter the waste tank 11 from the outlet of the second microchannel 5. Under the action of an electric field, the exosomes with the size of 30-200nm are driven to the position of the nano-pore film 12 to be captured, and free biological small molecules and other small vesicles pass through the nano-pore film 12 and enter the enrichment tank 10, and after electrophoresis, the exosomes can be removed from the recovery tank 9, so that the exosomes are separated and purified.
The purification method of the invention comprises the following steps: adding an equal volume of a first sodium chloride solution (16% (w/v) polyethylene glycol 6000,1M sodium chloride) containing polyethylene glycol 6000 into the supernatant containing the exosomes, standing for more than 12 hours, centrifuging 3500g for 30min, collecting the precipitate, re-suspending in a PBS buffer, adding an equal volume of a second sodium chloride solution (10% (w/v) polyethylene glycol 6000,1M sodium chloride) containing polyethylene glycol 6000 again, standing for more than 4 hours, centrifuging 3500g for 30min, collecting the precipitate, re-suspending in the PBS buffer, and obtaining the purified exosomes.
The invention also carries out diameter measurement on the obtained adipose-derived mesenchymal stem cell exosomes, the invention has no special limit on the measurement method, and the measurement method can be adopted by methods well known in the art, such as a transmission electron microscope or a nanoparticle analysis system, and the like, so that the diameter of the mesenchymal stem cell exosomes prepared by the invention is measured within the range of 55-100nm, and the average diameter is 63.65nm.
In another aspect, the invention also provides the use of such adipose mesenchymal stem cell exosomes and conjugated polypeptides as compositions in cosmetics, mainly comprising the following components: mesenchymal stem cell exosomes, TH peptide and palmitoyl tetrapeptide-7 coupled polypeptide, olive powder, red grape powder, tree peony bark extract, emulsifying agent, humectant, thickening agent, chelating agent and the balance of solvent.
The weight ratio of the exosome to the coupled polypeptide to the plant components in the above proportion is 1:2-3:3, the weight ratio of exosomes to conjugated polypeptide is 1:3, olive fruits in the plant components: red grape powder: the weight ratio of cortex moutan 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 of the anti-aging, anti-wrinkle, antioxidant and repairing regenerative cosmetics to the skin; (2) The application in cosmetics for promoting skin scar and wound healing; (3) application in antiallergic and anti-inflammatory cosmetics.
In addition, the formulation of the cosmetic may be selected in any of the following forms: A. mesenchymal stem cell exosomes are used as additives: an additive comprising the mesenchymal stem cell exosomes as an active ingredient; b: lyophilized powder formulation: freeze-drying and dormancy and freeze-drying the exosome concentrated solution to prepare a dry powder preparation. In embodiments of the present invention, additives containing ADSC exosomes may be prepared to prepare a variety of cosmetics, such as facial masks, creams, lotions, essences, eye creams, stock solutions, and the like.
Example 1:
referring to fig. 1, the specific operation method of the microfluidic chip includes: (1) Opening a bottom cover of the chip, attaching a completely matched common adhesive tape at the two horizontal channel windows, and sealing by the bottom cover; (2) Accurately weighing 0.1g agarose gel in a conical flask, adding 10mL of 1 xTAE buffer solution, heating and dissolving in a microwave oven to prepare agarose gel with the concentration of 1.0%, injecting the agarose gel into a chip from the positive electrode of an electrophoresis tank to completely fill a first micro-channel of the chip, placing the chip at the temperature of 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 micro-channel and a third micro-channel, wherein the second micro-channel is connected with a microinjection pump, and the third micro-channel is used for enriching exosomes; (3) Injecting PBS eluent with pH value of 6.0 into the chip from the second sample inlet and making the chip enter an enrichment pool below the nanopore membrane; (4) Taking about 80 mu L of human ADSC, filling the human ADSC into an injector, connecting the injector to a micro injection pump, filling 1 xTAE buffer solution into the anode of an electrophoresis tank and the cathode of the electrophoresis tank, adjusting the voltage of the electrophoresis tank to be 100-150V, and opening the injection pump while applying the voltage to enable an exosome in the human ADSC to directionally move under the action of an electric field, and capturing the exosome by a nano-pore film through agarose gel, thereby completing the extraction of the exosome. And the diameter of the extracted exosomes was measured, and the result showed a particle size in the range of 55-100nm, preferably 55-65nm, see fig. 2.
Example 2:
the preparation method comprises the steps of using 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 fruit powder, 2-6 parts of red grape powder, 2-6 parts of tree peony bark extract, 0-1 part of glyceryl stearate, 0-1 part of cetyl stearyl alcohol, 1-2 parts of urea, 0-1 part of glycerin, 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 for preparing the face cream.
The intermediate stem cell exosome can be added in the form of freeze-dried powder, and the specific operation method is that the purified exosome concentrated solution or the exosome concentrated solution frozen at the temperature of minus 80 ℃ is melted in water bath, freeze-dried excipient is added for dissolution, and the mixture is filtered and then placed in a freeze dryer for freeze-drying treatment, wherein the freeze-drying vacuum degree is maintained at 0.04mbar, and the freeze-drying time is 10-20h.
Example 3: anti-aging test using the cream of example 2
Aging of human skin is a complex process influenced by many factors, and it is considered that aging damage of skin is mainly caused by accumulation of free radicals, while SOD is the only metalloenzyme taking the radical of sun flowers as a substrate in the body, and can effectively remove the free radicals, so that the level of SOD is used as an index of resisting aging.
Female mice were selected for 60 random groups of 3: normal control group, invention group and market product group, wherein the market product is the same kind of anti-aging product from normal market, normal control group is filled with normal saline, and other animals in each group are filled with the product and the market product, after last administration for 1h, the orbit of the mouse is taken out blood, centrifugated, and the supernatant is taken out to measure SOD value.
TABLE 1 anti-aging results of cosmetics prepared according to the present invention
Group of SOD value (U/mg)
Normal control group 138.85±12.44
The invention group 159.63±15.36 **
Commercial product group 140.25±13.57 *
As can be seen from the above table, the SOD levels of the inventive and commercial products were significantly increased (P < 0.01) compared to the control group, while the difference between the inventive and commercial products was significantly (P < 0.05), the SOD values of the inventive group were significantly higher than the commercial group, indicating that the inventive group products could significantly increase the SOD levels.
Example 4: scar test Using the cream of example 2
By adopting the skin scar test method in the invention patent CN 110123838A, 7-8 week old female C57BL/6 mice are randomly divided into a control group, a skin scar model group and a treatment group, 10 mice in each group are subjected to dermis thickness measurement, and 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 and P < 0.05), so that the treatment group can prevent scar formation.
Example 5: anti-sensitization test Using the creams of example 2
The mice of the male and female halves are randomly divided into four groups, 12 mice in each group are respectively used as a normal control group, the second group is used as a positive control group, 1% DNCB is used for induction, then 1% OX is used for enhanced induction, the induction is repeated to sensitize the mice, the third group and the fourth group are used as treatment groups, wherein the third group is treated by using compound phellodendron lotion, the fourth group is treated by using the face cream provided by the invention, the skin of the mice in the normal control group is smooth, erythema, exudation and desquamation are avoided, the skin tissue state of the mice in the positive control group is obviously improved, the experimental effect of the group is not greatly different from that of the mice treated by drugs, the results are shown in table 2, the IGE level of the positive control group is obviously increased compared with the normal control group, the IGE level of the positive control group is reduced on average, and the face cream provided by the invention has no statistical difference with the IGE level treated by the drugs.
Table 2 shows the results of the anti-sensitization test of the cosmetics prepared according to the present invention

Claims (5)

1. Application of mesenchymal stem cell exosomes and compositions thereof in preparing cosmetics is characterized in that: the cosmetic comprises the following components: mesenchymal stem cell exosomes, TH peptide and palmitoyl tetrapeptide-7 conjugate, olive powder, red grape powder, tree peony bark extract, emulsifying agent, humectant, preservative, thickening agent, chelating agent and the balance of solvent;
the cosmetic comprises one or more of the following uses:
(1) Application in skin anti-aging cosmetics;
(2) The application in the cosmetics for preventing scar;
(3) Application in antiallergic cosmetic;
the mesenchymal stem cell exosomes and the compositions thereof comprise conjugated polypeptides, and the molecular structural formula of the conjugates is as follows:
the preparation method of the conjugate comprises the following steps:
synthesizing TH peptide by using a polypeptide solid-phase synthesis method;
dissolving TH peptide and palmitoyl tetrapeptide-7 in a methanol solvent according to a molar ratio of 1:1.2, and performing coupling under the protection of a catalyst and inert gas to ensure that the N-terminal amino group of the TH peptide and the carboxyl terminal of the palmitoyl tetrapeptide-7 form an amide bond to be connected, so as to prepare a TH peptide and palmitoyl tetrapeptide-7 conjugate;
the preparation method of the mesenchymal stem cell exosome comprises the following steps:
TRADD modified mesenchymal stem cells: constructing TRADD over-expression adenovirus, and transfecting mesenchymal stem cells by using the obtained TRADD over-expression adenovirus;
mesenchymal stem cell culture: culturing 3-5 generations of mesenchymal stem cells over-expressed by TRADD by using a low-sugar DMEM culture medium containing 10% fetal calf serum by volume concentration, culturing for 48 hours by changing a serum-free culture medium when the cells are fused to 70% -80%, and collecting supernatant after culturing;
separating and purifying the mesenchymal stem cell exosomes by a microfluidic technology to obtain the TRADD modified mesenchymal stem cell exosomes; the mesenchymal stem cells are adipose-derived mesenchymal stem cells;
the microfluidic technology separation mesenchymal stem cell exosome mainly adopts the microfluidic chip, the microfluidic chip comprises PDMS (polydimethylsiloxane) substrate and PC (polycarbonate) diaphragm, be equipped with the bottom on the diaphragm, the bottom is also the PC material, substrate, diaphragm pass through sticky tape sealed laminating with the bottom and be connected, be equipped with inlet, curved passageway, microchannel and liquid outlet on the diaphragm, wherein first inlet (4) and second inlet (7) are connected second microchannel (5) and third microchannel (8) respectively through curved passageway (6), second microchannel (5) and third microchannel (8) are horizontal mutual parallel arrangement, the other end of second microchannel (5) is connected abandonment groove (11) through the liquid outlet, the other end of third microchannel (8) is connected recovery groove (9) through the liquid outlet, second microchannel (5) and third microchannel (8) are perpendicular department still to be equipped with first microchannel (3), first microchannel (3) are equipped with respectively electrophoresis groove (1) and third microchannel (12) and electrophoresis groove (12) of negative electrode (12) are equipped with respectively.
2. Use of the mesenchymal stem cell exosomes and their compositions in the preparation of cosmetics according to claim 1, characterized in that: the micro-injection pump is connected with the first sample inlet (4) and the second sample inlet (7), the flow speed 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.
3. Use of the mesenchymal stem cell exosomes and their compositions in the preparation of cosmetics according to claim 1, characterized in that: the length of the first micro-channel (3) is 55mm, the width is 5mm, the depth is 100 mu m, the length of the second micro-channel (5) and the third micro-channel (8) is 48mm, the width is 2mm, the depth is 100 mu m, the size of the nano-pore film (12) is 5mm x 2mm, and the aperture of the nano-pore film (12) is 30-200nm.
4. Use of the mesenchymal stem cell exosomes and their compositions in the preparation of cosmetics according to claim 1, characterized in that: the diameter of the prepared mesenchymal stem cell exosome is within the range of 55-100 nm.
5. Use of the mesenchymal stem cell exosomes and their compositions in the preparation of cosmetics according to claim 1, characterized in that: the formulation of the cosmetic may be in any of the following forms:
mesenchymal stem cell exosomes are used as additives: an additive comprising the mesenchymal stem cell exosomes as an active ingredient;
lyophilized powder formulation: freeze-drying and dormancy and freeze-drying the exosome concentrated solution to prepare a dry powder preparation.
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