CN115232786A

CN115232786A - Adipose-derived stem cell exosome and application thereof

Info

Publication number: CN115232786A
Application number: CN202210923656.5A
Authority: CN
Inventors: 商婷; 韩昊天; 崔磊
Original assignee: Moisen Xiamen Shengfa Co ltd
Current assignee: Panas Cell Xiamen Biotechnology Co ltd
Priority date: 2022-08-02
Filing date: 2022-08-02
Publication date: 2022-10-25
Anticipated expiration: 2042-08-02
Also published as: CN115232786B

Abstract

The application discloses application of adipose-derived stem cell exosomes in preparation of a product for promoting hair follicle growth. The application also discloses an adipose-derived stem cell exosome suitable for promoting hair follicle growth, wherein the adipose-derived stem cell exosome is derived from adipose-derived stem cells and/or adipogenic induced adipose-derived stem cells. The application also discloses a pharmaceutical composition for promoting hair growth, which contains the adipose-derived stem cell exosome and a conventional pharmaceutical carrier.

Description

Adipose-derived stem cell exosome and application thereof

Technical Field

The specification relates to the field of medical cosmetology, in particular to an adipose-derived stem cell exosome.

Background

With the increasing pace of life, more and more people are affected by alopecia. Alopecia can negatively affect the confidence and self-esteem of people, thereby affecting mental health and even living conditions. Alopecia is a common disease of dermatology, has high prevalence rate and often affects physical and psychological health of patients. The existing treatment means mainly comprise modes of medicines, laser, operations and the like, but the methods face the defects of long treatment period, side effects of medicines, high cost, creativity and the like, so that the alopecia treatment is always an international difficult point and a hot point problem, and how to promote the hair regeneration is an urgent problem to be solved in the academic world. Adipose-derived stem cells (ADSCs) are one of the sources of adult progenitor cells, are isolated and extracted from Adipose tissues, have stromal cells with the same multipotentiality as bone marrow mesenchymal stem cells and peripheral blood stem cells, and are promising stem cell sources due to relative abundance, easy isolation, few complications of donor sites, strong self-renewal capacity, high proliferation potency, rapid expansion and low immunogenicity. The ADSCs can paracrine a plurality of growth factors, which are the key for the ADSCs to play a role in treatment, the growth factors have a treatment role, can participate in cell proliferation and apoptosis, immunoregulation, angiogenesis and inflammatory reaction, are widely applied in tissue repair and regeneration medicine, and are expected to become one of ideal methods for improving skin aging based on tissue regeneration. It has been demonstrated that local injection of adipose stem cells can promote hair follicle growth and allow the resting stage hair follicles to enter the anagen phase. The adipose-derived stem cells have strong adipogenic differentiation capacity. However, stem cell therapy has problems of long cell expansion time, difficulty in accurately matching cell growth with injection therapy, short survival time of cells in vivo, and the like.

Exosomes (exosomes) are extracellular vesicles with a diameter of 30-150nm, are membrane vesicle-like bodies released outside cells by fusion of a multivesicular body and a cell membrane, have cytoplasm and membrane components of the source cells, contain various components such as proteins, nucleic acids and lipids, are important carriers for cell-cell communication, and can release exosomes almost from all cells. The exosome belongs to a cell-free component, can be secreted into an extracellular environment by cells, is more stable and easy to store and transport, and can avoid the problems of immunological rejection, tumorigenesis and thrombosis caused by cell therapy. Has great clinical research prospect in disease treatment. None of the prior art discloses the association of exosomes with hair follicles.

Disclosure of Invention

According to an aspect of the present application, there is provided the use of an adipose stem cell exosome in the preparation of a product for promoting hair follicle growth.

According to another aspect of the present application, there is provided an adipose stem cell exosome suitable for promoting hair follicle growth, the adipose stem cell exosome being derived from adipose stem cells and/or adipogenic-induced adipose stem cells.

According to still another aspect of the present application, there is provided a pharmaceutical composition for promoting hair growth, comprising the adipose stem cell exosomes described above and a conventional pharmaceutical carrier.

The adipose-derived stem cell exosome and the application thereof disclosed by the application bring beneficial effects including but not limited to: (1) Exosomes belong to the cell-free components that can reduce or avoid the problems of immune rejection, tumorigenesis and thrombosis caused by cell therapy. (2) Exosomes are cell-free components, and are easy to store, transport, mass produce and convert into products. (3) In a preferred embodiment of the present application, the exosome is derived from an adipogenic-induced adipose-derived stem cell, and the capacity of the adipogenic-induced adipose-derived stem cell to secrete the exosome is stronger. (4) The exosome of the adipogenic induction fat stem cell can promote the growth of hair follicle cells, thus fundamentally solving the reason of alopecia. (5) Does not need to take medicines, and reduces the side effect of the medicines on the kidney and the liver.

Drawings

The present application will be further explained by way of exemplary embodiments, which will be described in detail by way of the accompanying drawings. These embodiments are not limiting, wherein:

FIG. 1 is a graph of adipogenic differentiation characteristics of adipose stem cells, according to some embodiments herein;

FIG. 2 is a fluorescence image showing detection of Mycoplasma contamination according to some embodiments of the present application;

FIG. 3 is a transmission electron micrograph of exosomes according to some embodiments of the present application;

FIG. 4 is a graph of a western blot identifying exosome-tagged proteins according to some embodiments of the present application;

FIG. 5 is a graph of particle size analysis of exosomes shown according to some embodiments of the present application;

FIG. 6 is a graph showing the results of adipose stem cells promoting hair growth in mice according to some embodiments of the present application;

fig. 7 is a comparative plot of pigmentation scores, according to some embodiments of the present application.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings used in the description of the embodiments will be briefly described below. It is obvious that the drawings in the following description are only examples or embodiments of the present description, and that for a person skilled in the art, the present description can also be applied to other similar scenarios on the basis of these drawings without inventive effort. Unless otherwise apparent from the context, or otherwise indicated, like reference numbers in the figures refer to the same structure or operation.

As used in this specification and the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that steps and elements are included which are explicitly identified, that the steps and elements do not form an exclusive list, and that a method or apparatus may include other steps or elements.

Certain sequences of written description are used in this specification to describe operations performed by systems according to embodiments of the specification. It should be understood that the preceding or following operations are not necessarily performed in the exact order written. Rather, the various steps may be processed in reverse order or simultaneously, as the case may be. Meanwhile, other operations may be added to the processes, or a certain step or several steps of operations may be removed from the processes.

Exosomes (exosomes) are miniature plasma membrane vesicle structures with a diameter of about 30-150nm actively synthesized by endoplasmic reticulum and golgi complex of intracellular plasma membrane organelles and secreted to the extracellular environment, are important paracrine forms outside cell soluble cytokines, have cytoplasmic and lipid membrane components of source cells, and contain source cell-specific proteins and related proteins such as CD63, CD9 and CD81. The exosome component mainly comprises: the biological active substances such as microRNA, mRNA and cytokines have important biological functions of reducing apoptosis, relieving inflammatory reaction, promoting angiogenesis, inhibiting fibrosis, improving tissue repair potential and the like. The exosome can be ingested into cells through endocytosis, so that the problem that macromolecules are not easily absorbed by the cells due to the blockage of cell membrane channels is avoided, and the exosome has the advantages of high biological activity and high action efficiency.

The research of the application shows that the adipose-derived stem cell exosome has the function of promoting hair follicle proliferation. When the hair follicle enters the anagen phase from the telogen phase, the growth and development of the hair follicle are played an important role along with the proliferation of dermal adipose tissue. The hair follicle regeneration needs fat cells to secrete special nutrient substances, and the exosome is extracted by inducing the fat stem cells into the fat cells and is discovered to achieve the effect of promoting the growth of the hair follicle through local injection.

The adipose stem cell exosomes of the present application may be derived from adipose stem cells. To increase the adipose stem cell exosome yield, in some embodiments, adipose stem cells may be induced to adipogenic-induced adipose stem cells. In some embodiments, the adipose stem cell exosomes may be derived from adipogenic-induced adipose stem cells.

Certain cytokines and hormones have concentration and time effects on the adipogenic differentiation of adipose-derived stem cells, i.e., may exert a bidirectional effect of promoting or inhibiting adipogenic differentiation depending on the concentration and action on the adipogenic differentiation stage. In some embodiments, the adipogenic-induced adipose stem cells can be obtained by inducing and culturing the adipose stem cells by using an adipogenic-induced medium. The existing fat stem cell adipogenesis induction methods are all suitable for the application. The adipogenic induction medium can be added with differentiation induction factors on the basis of a basic medium. The differentiation-inducing factor may be selected from: hormones, cytokines, etc., for example, may be selected from one or more of dexamethasone, insulin, isobutylmethylxanthine or indomethacin. In one embodiment of the present application, the final concentration of each stem cell differentiation promoting component is as follows: dexamethasone 0.1 μ M, insulin 10 μ g/ml, isobutylmethylxanthine (IBMX) 0.1mM, indomethacin 100 μ M. In some embodiments, adipose stem cells may develop adipogenic differentiation characteristics 7 days after induction culture.

The adipogenic induction fat stem cell should have adipogenic differentiation characteristics, such as the appearance of lipid droplets in oil red O staining, and the expression of adipogenic related genes and proteins such as PPAR gamma, LPL, fatty acid binding protein 4, activator protein 2, leptin, glucose transporter 4, and the like. In some embodiments, the adipose stem cell exosomes may comprise exosome marker proteins CD63 and CD81.

When adipose-derived stem cells are cultured using a serum-containing medium, since many foreign proteins are contained in the serum, foreign proteins are easily introduced into exosomes. In order to remove foreign proteins, one of the embodiments of the present specification provides a method for preparing the above-described adipose-derived stem cell exosome. In some embodiments, the cells may be cultured in serum-free medium for 24h to 48h after induction of differentiation. The serum-free medium may be a conventional serum-free medium, to which antibiotics and serum substitutes may be added at appropriate concentrations. In some embodiments, the serum-free medium can be a medium containing EGM-2MV, 1 serum replacement, and 1% dual streptomycin antibody.

In order to remove impurities such as cell debris, in some embodiments, the supernatant may be collected, centrifuged, and discarded to remove impurities from the supernatant. In some embodiments, the exosomes may be resuspended for multiple washes of exosomes. In some embodiments, the exosomes may be resuspended using PBS or other phosphate buffer.

To reduce the loss of exosomes, long term high speed centrifugation can be used. In some embodiments, the time of centrifugation may be 60min to 80min. In some embodiments, the time for centrifugation may be 70min, which is preferred. In some embodiments, the rotational speed of the centrifugation can be 10000g to 100000g.

When centrifuging at high speed for a long time, the temperature in the centrifuge rises with time, so that it is necessary to operate this step using a centrifuge capable of controlling the temperature. In some embodiments, the temperature of centrifugation may be between 2 ℃ and 6 ℃. In some embodiments, the temperature of centrifugation may be 4 ℃ as preferred.

The exosomes contain proteins, and in order to prevent protein degradation, in some embodiments, the exosomes may be stored in a refrigerator at-80 ℃.

One embodiment of the specification further provides application of the adipose-derived stem cell exosome in preparation of a product for promoting hair follicle growth. Illustratively, products that promote hair follicle growth are used to prevent hair follicle loss, prevent hair follicle damage, and promote hair follicle growth. In some embodiments, the application may be promoting hair growth.

One embodiment of the specification also provides a pharmaceutical composition for promoting hair growth. In some embodiments, the pharmaceutical composition may comprise the adipose stem cell exosomes described above and a conventional pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may contain adipogenic-induced adipose stem cell exosomes. In some embodiments, the pharmaceutical composition may be an injection, tablet, capsule, microparticle, ointment, or the like.

In some embodiments, the pharmaceutically acceptable carrier may include pharmaceutically acceptable emulsion components, cream components, injection solutions. In some embodiments, the pharmaceutically acceptable carrier may be one or more of high concentration platelet plasma, keratin, or hyaluronic acid. In some embodiments, the pharmaceutical carrier may include excipients or lyoprotectants. The adjuvants include mineral oil, stearic acid, palmitic acid, glyceryl stearate, shea butter, myristyl myristate, hydrogenated polydecene, hydrogenated polyisobutene, trimethylsiloxysilicate, polydimethylsiloxane, behenyl alcohol, caprylic/capric triglyceride glycerol, panthenol, etc.; or butanediol, dipropylene glycol, sorbitol, glycerol, sodium hyaluronate, trehalose, ceramide, amino acid, sorbitol, betaine, polyethylene glycol-6, etc. suitable for making facial mask; can also be prepared into injection by sodium hyaluronate, chitosan, collagen or normal saline, etc.; it can also be lyophilized protectant such as trehalose, mannitol, dextran, polyethylene glycol, amino acid, etc. which can be directly applied to skin.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1

The test steps are as follows:

1. preparing adipose-derived stem cells and adipogenic differentiated adipose-derived stem cells:

taking fat in the inguinal region of the mouse, carefully separating the fat by using forceps, placing the separated fat into 20ml of PBS, cutting off obvious blood vessels, cleaning the blood vessels for 1 time by using sterile PBS, transferring the blood vessels to a sterile dish cover, and cutting the blood vessels to be chyle by using sterile scissors.

Digestion: cutting 1ml of gun head (enlarging opening), absorbing collagenase, putting into a dish cover, putting into 4-5ml of 0.075-percent type I collagenase together with chyliform fat, carrying out shake digestion at 37 ℃ and 120rpm for 10-15min, then carrying out centrifugation at 2000rpm for 5min, discarding upper fat and supernatant, carrying out re-suspension precipitation by using culture solution, inoculating into a 6cm culture dish for culture (3 ml of culture medium DMEM +10 FBS +1% streptomycin), and carrying out growth and passage to P3.

The 3 rd generation ADSCs are inoculated into a 10cm culture dish, when the cells grow to 80%, a adipogenic induction culture medium (containing 0.1 mu M of dexamethasone, 10 mu g/ml of insulin, 0.1mM of isobutyl methylxanthine (IBMX) and 100 mu M of indomethacin) is added for culture, and the culture solution is changed once every 3 days.

2. Oil red staining was performed 7 days after induction:

preparing an oil red O dyeing working solution according to the proportion of oil red O isopropanol saturated solution to distilled water = 3;

the cells in the dish were washed once with PBS and fixed in 4% paraformaldehyde at 4 ℃ for 15min.

Washing with PBS:

adding 500 mul of oil red O staining working solution into each hole of a six-hole plate, covering, dip-staining for 10-15 minutes at room temperature, observing the staining of lipid drops under a microscope, and washing with PBS.

And (3) decoloring:

sucking PBS, slightly drying in the air (drying in the air and immediately performing subsequent operations), adding equal amount of isopropanol into each hole in the biological safety cabinet, sucking out the isopropanol, and removing excessive dye.

And (4) sealing the glycerol gelatin (the glycerol gelatin can be stored for a long time after sealing).

Microscopic observation and photography showed that grape-like red lipid droplets were visible in microscopic observation as shown in FIG. 1, indicating that the cultured adipose stem cells had differentiated into adipogenic-induced adipose stem cells.

3. Detecting adipogenic differentiation indexes:

washing cells in the culture plate for 2 times by using PBS, removing the PBS, adding 1ml of TransZol Up into each hole to lyse the cells, repeatedly blowing the cells by using a pipette, transferring the lysate to a 1.5ml EP tube, and standing for 5 minutes at room temperature;

adding 0.2ml of chloroform into each tube, violently shaking for 30 seconds, and incubating for 3 minutes at room temperature;

centrifuging at 12000rpm at 4 ℃ for 15 minutes; after centrifugation, the mixed liquid is divided into three layers, namely a colorless aqueous phase (upper layer), a middle layer and a pink organic phase (lower layer), wherein RNA is in the colorless aqueous phase at the upper layer;

transferring the upper colorless aqueous phase into a new centrifugal tube without RNase, and taking care that the gun head does not adhere to the wall when sucking the water and does not suck the intermediate phase;

adding 500 mul of isopropanol into each tube, reversing and uniformly mixing, and incubating for 10 minutes at room temperature;

centrifuging at 12000rpm at 4 ℃ for 10 minutes to form a gelatinous precipitate on the bottom and the side wall of the tube;

discard the supernatant, add 1ml of 75% ethanol (prepared with 100% ethanol and DEPC treated water) per tube, vortex vigorously;

7500rpm, 5 minutes at 4 ℃;

discarding the supernatant, centrifuging by a palm centrifuge for a short time to throw off ethanol on the tube wall, carefully absorbing most ethanol solution at the tube bottom, and drying RNA precipitate in air at room temperature for about 5 minutes until white precipitate becomes semitransparent; dissolving RNA precipitation: each tube was filled with 20. Mu.l of RNase-free water and blown with a gun several times to dissolve completely.

Measuring the concentration, namely measuring the concentration of the RNA solution by using an ultramicro ultraviolet-visible spectrophotometer;

carrying out reverse transcription according to the operation instruction of a 5x All-In-One RT MasterMix reverse transcription kit;

the obtained cDNA is subjected to fluorescent quantitative PCR, and the expression of genes C/EBP beta, adiposin and LPL is obvious when the adipose-derived stem cells are differentiated into adipogenic induced adipose-derived stem cells.

4. Detecting the presence of bacteria and mycoplasma in the culture solution:

culturing cells on a cover glass; cells at 70% confluence were used for mycoplasma detection.

Rinsing the cover glass with Hanks' solution without phenol red;

adding 5ml of fixing solution, and standing for 10min;

rinsing with deionized water;

5ml of working solution of dibenzoamide fluorescent dye (Hoechst 33258) is used for dyeing for 10min;

the cover glass is dried in the air, the cells face upwards, a plurality of drops of phosphate buffer solution with the pH value of 5.5 are dripped, and the observation is carried out under a fluorescence microscope, the result is shown in figure 2, the staining on the cell surface is avoided, the sample is free from mycoplasma pollution, and the cover glass can be used for subsequent experiments.

5. Extraction of exosomes:

when the cell fusion degree cultured in the step 1 reaches about 80%, the culture medium is sucked away, washed for 2 times by PBS, then replaced by a serum-free culture medium (EGM-2MV +1x serum substitute +1% streptomycin double antibody), and cultured for 24-48h to collect the supernatant.

The supernatant was centrifuged (at 4 ℃) as follows:

300g,10min;2000g,10min;10000g,30min, and the supernatant is retained and filtered by a 0.22um filter head.

Collecting exosomes by an ultracentrifugation method: (ultracentrifuge process)

Centrifuging at 100000g in an ultracentrifuge at 4 deg.C for 70min, discarding supernatant, and suspending the exosome in a centrifuge bottle.

Centrifuging at 100000g in ultracentrifuge at 4 deg.C for 70min, discarding supernatant, resuspending exosome with 200ul PBS, taking small amount, measuring concentration, and storing in refrigerator at-80 deg.C.

Example 2

Morphology of the adipogenic-induced adipose stem cell exosomes obtained in example 1 was observed by Transmission Electron Microscopy (TEM), and the result is shown in fig. 3. As can be seen by observation under an electron microscope, the exosomes obtained by the extraction method all present typical forms of exosomes and are round or oval vesicle-shaped structures.

Example 3

Westernblot detection of the expression of exosomes:

a sample was prepared by preparing a lysis buffer consisting of 50mM Tris, pH7.4,40mM NaCl,1mM EDTA,0.5% Triton X-100,50mM NaF,10mM sodium pyrophosphate, 10mM sodium glycerophosphate and a mixture of the corresponding protease inhibitor and phosphatase inhibitor. The lysis buffer was added to the adipogenic-induced adipose stem cell exosomes ADSC-Exos obtained in example 1 and lysed on ice for 30min, collected into 1.5ml EP tubes, centrifuged at 10,000rpm,10min and the supernatant was collected. The concentration of ADSC-Exos protein was determined using the BCA protein quantification kit as described above according to the protocol. The ADSC-Exos samples were then mixed with loading buffer (5X), heated at 100 ℃ for 10min to denature the proteins, and stored at-80 ℃.

And (3) electrophoresis, namely preparing SDS-PAGE gel, filling the prepared gel into an electrophoresis tank, pouring electrophoresis buffer solution into the electrophoresis tank from the inside and the outside, and adding a marker and a sample into a sample loading hole by using a micropipette, wherein each hole is added with 30 mu g of the sample. After 80V of constant voltage is run to the separation gel, the voltage is adjusted to 120V, protein marker is observed, and electrophoresis is stopped after protein strips are separated.

And (3) transferring the membrane, namely cutting the PVDF membrane into the size of 6.6x8.5cm, activating in methanol for 5min, and placing in a membrane transferring buffer solution for later use. The gel that has been electrophoresed is removed and the electrophoresis solution is rinsed off with water. The transfer system was installed in the order of cathode clamping plate → sponge → filter paper → gel → PVDF film → filter paper → sponge → anode clamping plate. And (3) placing the membrane rotating device in a foam ice box, adding a membrane rotating buffer solution (the gel is close to the negative electrode, and the PVDF membrane is close to the positive electrode), and rotating the membrane for 1.5 hours at a voltage of 100V.

Sealing, namely soaking the PVDF membrane in 5 percent of skim milk for sealing after the membrane is converted. 5% skim milk was prepared with 1xTBST, and TBST was prepared with 500. Mu.l Tween 20 in 500ml 1xTBS, and the PVDF membrane was then placed in 5% skim milk and blocked on a shaker at room temperature for 1h.

Primary anti-incubation, removing the blocking solution after blocking is finished, washing the membrane with 1xTBST for 5 min/time, and 5 times in total. TSG101 (1 dilution, abcam ab 125011), CD9 (1.

Secondary antibody incubation primary antibody was recovered and the membrane washed again with 1xTBST (5 min/time, 5 times total), then secondary antibody was diluted at 1.

And (3) chemiluminescence and development, namely exposing and developing through a chemiluminescence system, recording the information of the sample, the sequence and the antibody in detail, and storing the data result.

The results are shown in fig. 4, which illustrates that the adipogenic-induced adipose-derived stem cells express the exosome marker proteins CD63 and CD81, and further illustrates that the lipogenic-induced adipose-derived stem cells are identified as adipose-derived stem cell exosomes.

Example 4

The Particle diameter of the adipogenic-induced adipose stem cell exosomes obtained in example 1 was examined with ZetaView PMX 110 (Particle Metrix, germany).

Washing a sample pool by using deionized water, and calibrating a ZetaView system by using polystyrene microspheres (100 nm);

washing the sample cell with 1x PBS;

diluting the separated ADSC-Exos with 1x PBS, and detecting the sample;

11 points were recorded and analyzed, the temperature was maintained at 23-30 ℃;

the data were saved and plotted using Origin software to generate NTA particle size analysis curves, the results of which are shown in FIG. 5, and the results show that the mean size was about 175nm, which substantially corresponds to the exosome size.

Example 5

In vivo mouse experiments:

adult C57/6J mice were taken at 8 weeks, and the hair of the mice at this time period was in telogen. An electric hair pusher is used for pushing and polishing the hair with the area of 2cmx2cm on the back. Mice were divided into three groups: group A is control group, injected with 250 μ L PBS subcutaneously; group B, 250. Mu.L of adipose-derived stem cell exosomes were injected subcutaneously into the back (400. Mu.g/mouse, 50. Mu.L/injection site, total 5 injection sites); group C, dorsal subcutaneous injection of 250. Mu.L of the adipogenic-induced adipose stem cell exosomes prepared in example 1 (400. Mu.g/mouse, 50. Mu.L/injection site, total 5 injection sites); after 14 days, the state of hair regrowth was observed and photographed, and the results are shown in FIG. 6. As can be seen from fig. 6, the back hair in group C clearly exceeds the other two groups, indicating that the effect of adipogenic-induced adipose stem cell exosomes on promoting hair growth is significant.

Mice were sacrificed and dorsal skin was taken for H & E staining:

fixing: excised skin tissue was fixed with 4% paraformaldehyde.

And (3) dehydrating: placing fixed skin tissue into gradient alcohol for dehydration, 75% alcohol overnight, 85% alcohol for 2h,90% alcohol for 1h,95% alcohol for 1h,100% alcohol for 30min, and 100% alcohol for 30min.

And (3) transparency: sequentially placing skin tissue into xylene I for 10min, and xylene II for 5-10min.

Embedding: sequentially putting skin tissues into paraffin I, II and III, respectively soaking in paraffin for 1h, and trimming.

Slicing: the trimmed paraffin blocks were placed on a microtome and sliced successively, each slice being 4 μm thick.

Spreading: and floating the cut paraffin sections on the surface of warm water at 40 ℃, flattening, and fishing out the paraffin sections by using the anti-falling glass slide.

Baking slices: and (3) placing the glass slide in an oven at 60 ℃ for baking for 2-3h.

Dewaxing, namely putting the slices into dimethylbenzene, and dewaxing for 15 minutes.

Rehydration, namely putting the slices into 100 percent, 95 percent, 85 percent and 75 percent ethanol in turn, wherein each gradient ethanol lasts for 5 minutes; distilled water for 1 minute.

Hematoxylin staining was performed for 8 minutes.

And (4) washing, namely washing the excessive loose color by using distilled water.

Differentiation, 1% ethanol hydrochloride differentiation for several seconds.

And returning blue, namely flushing for 10 minutes by running water.

Eosin staining for 1 min.

Washing with distilled water to remove loose color; dehydration 75%, 85%, 95%, 100% alcohol gradient dehydration, each gradient for 5 minutes.

And (4) transparent xylene for 10 minutes.

And (4) sealing, namely sealing the neutral gum.

Ventilating in a fume hood to disperse odor, observing under a microscope, and counting the number of hair follicles, wherein the result is shown in figure 7, after the fat-forming induced adipose-derived stem cell exosome is injected subcutaneously on the back of a mouse, compared with other two groups of mice, the number of hair follicles is remarkably increased, which indicates that the fat-forming induced adipose-derived stem cell exosome can remarkably improve the number of the hair follicles on the back of the mouse.

Having thus described the basic concept, it will be apparent to those skilled in the art that the foregoing detailed disclosure is to be considered as illustrative only and not limiting, of the present invention. Various modifications, improvements and adaptations to the present description may occur to those skilled in the art, though not explicitly described herein. Such modifications, improvements and adaptations are proposed in the present specification and thus fall within the spirit and scope of the exemplary embodiments of the present specification.

Also, the description uses specific words to describe embodiments of the description. Reference throughout this specification to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic described in connection with at least one embodiment of the specification is included. Therefore, it is emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, some features, structures, or characteristics of one or more embodiments of the specification may be combined as appropriate.

Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present disclosure. Other variations are also possible within the scope of the present description. Thus, by way of example, and not limitation, alternative configurations of the embodiments of the specification can be considered consistent with the teachings of the specification. Accordingly, the embodiments of the present description are not limited to only those embodiments explicitly described and depicted herein.

Claims

1. Application of adipose-derived stem cell exosomes in preparation of products for promoting hair follicle growth.

2. The use of claim 1, wherein the adipose stem cell exosomes are derived from adipose stem cells and/or adipogenic-induced adipose stem cells.

3. The use of claim 2, wherein the adipogenic-induced adipose stem cells are derived from adipose stem cells by induction culture using an adipogenic induction medium.

4. The use of claim 2, wherein said adipogenic-induced adipose stem cells are characterized by adipogenic differentiation.

5. The use of claim 1, wherein said exosomes comprise exosome marker proteins CD63 and CD81.

6. An adipose stem cell exosome suitable for promoting hair follicle growth, derived from adipose stem cells and/or adipogenic-induced adipose stem cells.

7. An adipose-derived stem cell exosome according to claim 6, wherein the adipogenic induced adipose stem cell is obtained by inducing and culturing an adipose-derived stem cell using an adipogenic induction medium; and/or the adipose-derived stem cell exosomes contain exosome marker proteins CD63 and CD81; and/or, the adipogenic induced adipose stem cells have adipogenic differentiation characteristics.

8. The method for producing an adipose-derived stem cell exosome according to claim 6 or 7, comprising the following steps:

inoculating adipose-derived stem cells into a cell culture medium for cell culture;

adding a adipogenic induction culture medium to induce the adipogenic differentiation of the adipose-derived stem cells;

removing the culture medium, and replacing the serum-free culture medium for culture;

collecting supernatant, centrifuging, filtering and re-centrifuging to obtain the adipose-derived stem cell exosome.

9. The method for producing an adipose-derived stem cell exosome according to claim 8,

the adipogenesis induction culture medium is based on a basic culture medium and is added with differentiation induction factors; preferably, the differentiation inducing factor is selected from one or more of dexamethasone, insulin, isobutyl methylxanthine and indomethacin;

and/or, culturing for 24-48h after the serum-free culture medium is replaced, and collecting the supernatant.

10. A pharmaceutical composition for promoting hair growth, comprising the adipose stem cell exosome of claim 6 or 7 and a conventional pharmaceutical carrier.