Application of adipose stem cell exosomes in hair growth

Abstract

The present invention relates to a use of adipose stem cell exosomes in hair growth, and the preparation method of the adipose stem cell exosomes is as follows: take the 3rd to 6th generation of mouse adipose stem cells of logarithmic growth and culture them with complete culture fluid containing FBS until the cell density reaches 70%-80%, switch to DMEM culture fluid containing exosome-free FBS and continue to culture for 45-50 hours, collect the supernatant culture fluid, centrifuge to remove cells and cell debris, and then use a 100kDa molecular weight ultrafiltration tube to concentrate at 1500-2500g for 30min, add exosome rapid extraction reagent to the concentrated solution for extraction, centrifuge, and collect mouse adipose stem cell exosomes. In vitro and in vivo experiments show that the mouse adipose stem cell exosomes of the present invention can significantly promote hair growth with outstanding effects.

Description

Technical Field

[0001] The present invention relates to the medical application of stem cell exosomes, and in particular to the application of adipose stem cell exosomes in hair growth. Background Art

[0002] Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells that exist in adipose tissue and have multidirectional differentiation potential. In vitro and in vivo, ADSCs have the potential to differentiate into a variety of cell lines, including adipocytes, chondrocytes, osteoblasts, neurons, endothelial cells, and tendon cells.

[0003] Exosomes are a type of extracellular vesicles that can be secreted into the cell culture supernatant, have a lipid bilayer structure, and are 30-150nm in diameter. Exosomes contain a variety of proteins, nucleic acids (miRNA, mRNA, lncRNA, circRNA), and lipids, and are important carriers of intercellular communication. Almost all cells can release exosomes.

[0004] Exosomes have a wide range of applications and can be used to treat a variety of diseases with minimal side effects and reduced human rejection reactions.

[0005] Patent document CN112704688A discloses a hair growth injection based on autologous adipose stem cell exosomes, the hair growth injection is composed of autologous adipose stem cell exosomes and injection water, and its preparation method includes the following steps: step 1, selection of autologous fat; step 2, preparation of adipose stem cells; step 3, preparation of adipose stem cell exosomes; step 4, preparation of adipose stem cell exosome hair growth injection. Patent document CN110339338A discloses a hair growth agent, the hair growth agent comprises adipose mesenchymal stem cell conditioned medium, fibroblast exosomes, mannitol, trehalose and dextran 40, wherein the adipose mesenchymal stem cells are treated with 20-100mJ / cm 2 After irradiation with a UVB radiation dose of , the irradiated adipose mesenchymal stem cells secrete a variety of bioactive factors, which can stimulate the hair papilla and activate hair follicle stem cells, achieving the effect of hair growth from the inside out.

[0006] As we know, the extracellular environment is complex, and it is difficult for common exosome isolation methods to completely separate non-vesicle entities and extracellular vesicle components. Therefore, the exosome separation method affects the composition of the isolated extracellular vesicles, and thus produces functional differences. In addition, the type and state of cells secreting exosomes are also important factors affecting the function of exosomes. There is no report on mouse adipose stem cell exosomes and their use for hair growth as described in this application. Summary of the invention

[0007] The purpose of the present invention is to provide a use of mouse adipose stem cell exosomes in view of the deficiencies in the prior art.

[0008] In a first aspect, the present invention provides a use of mouse adipose stem cell exosomes in the preparation of a hair growth drug, wherein the mouse adipose stem cell exosomes are prepared according to the following method:

[0009] (a) Obtained logarithmically grown mouse adipose-derived stem cells from passages 3 to 6, cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%, aspirated the original culture medium, washed with PBS, replaced with DMEM culture medium containing 10% exosome-free FBS, and continued to culture in a 37°C, 5% CO2 incubator for 45-50 hours, and collected the supernatant culture medium;

[0010] (b) The supernatant culture medium was centrifuged at 3000 g for 15 min to remove cells and cell debris, and the supernatant was transferred to a 100 kDa molecular weight ultrafiltration tube and centrifuged again at 1500-2500 g for 30 min to collect the concentrate;

[0011] (c) Add the exosome extraction reagent to the concentrate, gently shake to mix, and place at 4°C for at least 12 h;

[0012] (d) The mixture was centrifuged at 4°C, 1500 g for 30 min, the supernatant was carefully discarded, PBS was added and the mixture was centrifuged again at 1500 g for 5 min, and the residual liquid was removed by aspiration to obtain the mouse adipose stem cell exosomes.

[0013] As a preferred example of the present invention, the mouse adipose stem cells are isolated from 5-10 day old C57BL / 6 mice.

[0014] As another preferred embodiment of the present invention, in step (a), the culture medium is changed to DMEM culture medium containing 10% exosome-free FBS and then culture is continued for 48 hours, and the supernatant culture medium is collected.

[0015] As another preferred embodiment of the present invention, in step (a), after the original culture medium is removed, the cells are washed with PBS for 1-3 times.

[0016] As another preferred embodiment of the present invention, the centrifugation parameters after the supernatant in step (b) is transferred to the ultrafiltration tube are: 1500g, 30min.

[0017] In a second aspect, the present invention provides a mouse adipose stem cell exosome, wherein the mouse adipose stem cell exosome is prepared according to the following method:

[0018] (a) Obtained logarithmically grown mouse adipose-derived stem cells from passages 3 to 6, cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%, aspirated the original culture medium, washed with PBS, replaced with DMEM culture medium containing 10% exosome-free FBS, and continued to culture in a 37°C, 5% CO2 incubator for 45-50 hours, and collected the supernatant culture medium;

[0019] (b) The supernatant culture medium was centrifuged at 3000 g for 15 min to remove cells and cell debris, and the supernatant was transferred to a 100 kDa molecular weight ultrafiltration tube and centrifuged again at 1500-2500 g for 30 min to collect the concentrate;

[0020] (c) Add the exosome extraction reagent to the concentrate, gently shake to mix, and place at 4°C for at least 12 h;

[0021] (d) The mixture was centrifuged at 4°C, 1500 g for 30 min, the supernatant was carefully discarded, PBS was added and the mixture was centrifuged again at 1500 g for 5 min, and the residual liquid was removed by aspiration to obtain the mouse adipose stem cell exosomes.

[0022] In a third aspect, the present invention provides the use of the mouse adipose stem cell exosomes as described above in the preparation of a drug for promoting an increase in the number of hair follicles and / or dermal thickness.

[0023] In a fourth aspect, the present invention provides the use of the mouse adipose stem cell exosomes as described above in preparing an experimental reagent for promoting the proliferation of hair follicle dermal papilla cells in vitro.

[0024] In a fifth aspect, the present invention provides a pharmaceutical composition for promoting hair growth, wherein the pharmaceutical composition contains the mouse adipose stem cell exosomes as described above and a conventional pharmaceutical carrier.

[0025] The advantages of the present invention are:

[0026] The present invention prepares a kind of mouse adipose stem cell exosomes by a fast and efficient method. In vitro and in vivo experiments confirm that the mouse adipose stem cell exosomes have a significant promoting effect on the proliferation of hair follicle papilla cells, can significantly increase the number of mouse hair follicles and the thickness of the dermis, and promote the growth of mouse hair. The effect is significantly stronger than that of exosomes prepared by other methods. Hair loss is a common disease in dermatology, with a high prevalence and often affecting the physical and mental health of patients. Existing treatment methods mainly include drugs, lasers, surgery and other methods, but these methods face the shortcomings of long treatment cycles, drug side effects, high costs and invasiveness. Therefore, hair loss treatment has always been a difficult and hot issue internationally. How to promote hair regeneration is a difficult problem that needs to be solved urgently in academia. The mouse adipose stem cell exosomes with significant effects on hair growth of the present invention are expected to be developed as drugs for treating hair loss, thereby providing a non-surgical, cell-free treatment method for hair loss treatment. Because exosomes are cell-free, they have the advantages of low immunogenicity, no tumorigenicity, and no thrombogenicity compared to current surgical treatments for hair loss; and compared to current drugs for treating hair loss, because exosomes contain nucleic acids (such as microRNA), proteins, and lipids that can participate in intercellular information exchange, these functional molecules may target effector cells of hair growth (such as hair follicle papilla cells) to exert their effects, thereby providing more precise treatment. In addition, from the perspective of operability, exosomes are cell-free and easy to store, transport, mass-produce, and convert into products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Isolated ADSC-Exo.

[0028] Figure 2 : Particle size analysis of ADSC-Exo.

[0029] Figure 3 : Transmission electron microscopy analysis of ADSC-Exo.

[0030] Figure 4 :Identification of exosome marker proteins by western blot.

[0031] Figure 5 :ADSC-Exo promotes the proliferation of hair follicle dermal papilla cells. *P<0.05, **P<0.01.

[0032] Figure 6 :ADSC-Exo promotes hair growth in mice. A. The hair growth of the two groups of mice was observed with naked eyes. B. The statistical results of the number of hair follicles and dermal thickness of the two groups of mice. *P<0.05, **P<0.01.

[0033] Figure 7 :The adipogenic differentiation of isolated and cultured adipose stem cells showed positive oil red staining.

[0034] Figure 8 :The adipose-derived stem cells isolated and cultured showed chondrogenic differentiation, showing positive cell aggregates stained with Alecin blue.

[0035] Fig. 9 :The osteogenic differentiation of isolated and cultured adipose stem cells showed positive results of Alizarin red staining. DETAILED DESCRIPTION

[0036] The specific implementation modes provided by the present invention are described in detail below with reference to the accompanying drawings.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] 1 Materials

[0040] 1.1 Animal sources

[0041] The experiment used SPF-grade C57BL / 6 male mice, which were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and kept in the Experimental Animal Center of Tongji University. The experimental research ethics were approved by the Experimental Animal Center of Tongji University.

[0042] 1.2 Main experimental reagents / consumables

[0043] DMEM culture medium (Gibco, USA, catalog number: C11995500BT), phosphate buffer solution (PBS, Gibco, USA), 0.05% trypsin solution (Trypsin-EDTA, Gibco, USA), Fetal Blood Serum (FBS, Gibco, USA, catalog number: 10099141C), and type I collagenase (Worthington, USA). Ultra-15 Centrifugal Filter Unit (Millipore, USA, catalog number UFC910008), ExoQuick-TC (catalog number EXOTC10A-1, SBI Biosciences, SBI, USA), Anti-CD9 antibody (abcam, UK), Anti-CD81 antibody (abcam, UK), Anti-TSG101 antibody (abcam, UK).

[0044] 2 Methods

[0045] 2.1 Preparation of Adipose-derived Stem Cells

[0046] (1) Adipose tissue was obtained from SPF-grade, 7-day-old C57BL / 6 mice. Bilateral inguinal fat was obtained under a sterile environment, and the blood vessels and fibrous components visible to the naked eye were removed.

[0047] (2) The adipose tissue specimens were repeatedly rinsed several times with a sterile PBS rinse solution containing 1% antibiotics (penicillin / streptomycin) to remove bloody fluid and fascia components and obtain pure adipose tissue.

[0048] (3) Use tissue scissors to cut the adipose tissue into chyme, then add 0.1% type I collagenase and digest at 37°C for 30 min to ensure uniform digestion of the tissue.

[0049] (4) Centrifuge at 2000 g for 10 min, discard the supernatant, add DMEM medium containing 10% fetal bovine serum, and place in a 37°C, 5% CO2 incubator for adherent culture.

[0050] (5) After 24 hours, change the medium, wash away the non-adherent cells and other impurities with PBS, then add fresh culture medium, and change the medium every three days. When the cell density reaches 80%, use 0.05% trypsin to digest and subculture. After obtaining the third generation of adipose stem cells, the cells are identified by detecting their potential for adipogenic differentiation, chondrogenic differentiation, and osteogenic differentiation. After 2-3 weeks of culture for adipogenic, chondrogenic, and osteogenic differentiation induction using adipogenic induction medium, chondrogenic induction medium, and osteogenic induction medium, respectively, oil red, alcian blue, and alizarin red staining were performed to observe the staining results. The results showed that the identified cells met the characteristics of adipose stem cells ( Figure 7-9 ).

[0051] 2.2 Preparation of Adipose-derived Stem Cell Exosomes

[0052] (1) The third generation of adipose-derived stem cells growing logarithmically were cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%. The original culture medium was aspirated, and the cells were washed three times with PBS. The cells were replaced with 10% exosome-free FBS DMEM culture medium and continued to be cultured in a 37°C, 5% CO2 incubator for 48 hours. The supernatant culture medium was collected.

[0053] (2) Centrifuge the supernatant at 3000 g for 15 min to remove cells and cell debris. Transfer the supernatant to a 100 kDa molecular weight ultrafiltration tube and centrifuge again at 1500 g for 30 min. Concentrate the supernatant to 1-2 ml and collect the concentrate.

[0054] (3) Add ExoQuick-TC (SBI) reagent to the concentrate at a volume ratio of 1:5, gently shake to mix, and place at 4°C for 12 h.

[0055] (4) Centrifuge the mixture at 4°C, 1500g for 30 min. Carefully discard the supernatant, add PBS and continue centrifugation at 1500g for 5 min, then remove the residual liquid. Add an appropriate amount of PBS or sterile water for injection, and gently blow the precipitate to obtain a resuspended solution.

[0056] (5) The exosome concentration was detected using a BCA protein assay kit, and the remaining exosomes were stored at -80°C for future use.

[0057] 2.3 Identification of exosomes from adipose-derived stem cells

[0058] Adipose stem cell exosomes were identified by using nanoparticle tracking analyzer (NTA), transmission electron microscopy (TEM) and western blot.

[0059] 2. Results

[0060] P3 ADSCs were cultured for 48 h under exosome-free serum conditions, and the supernatant was collected. Exoquick TC reagent was used to extract ADSC-Exo. Figure 1 ). Results ADSC-Exo were successfully extracted and identified by nanoparticle tracking analyzer (NTA), transmission electron microscopy (TEM) and western blot.

[0061] 1Nano-particle tracking analyzer (NTA)

[0062] The particle size of ADSC-Exo was analyzed by NTA method, and the results showed that its average size was about 144.3nm, which was basically consistent with the size of exosomes ( Figure 2 ).

[0063] 2 Transmission electron microscopy (TEM)

[0064] The morphology of ADSC-Exo was analyzed by transmission electron microscopy, and the results showed that the morphology of exosomes was present under electron microscopy ( Figure 3 ).

[0065] 3 western blot

[0066] The expression of three marker proteins TSG101, CD9, and CD81 of exosomes was identified by western blot. The results are as follows Figure 4 As shown, all three marker proteins were expressed in ADSC-Exo, further confirming that the identified cells were ADSC-Exo.

[0067] Example 2

[0068] 1. Materials and Methods

[0069] 1 Materials

[0070] 1.1 Animal sources

[0071] The experiments used SPF-grade C57BL / 6 male mice and SPF-grade SD male rats, which were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and kept in the Experimental Animal Center of Tongji University. The experimental research ethics were approved by the Experimental Animal Center of Tongji University.

[0072] 1.2 Main experimental reagents / consumables

[0073] DMEM culture medium (Gibco, USA, catalog number: C11995500BT), phosphate buffer solution (PBS, Gibco, USA), 0.05% trypsin solution (Trypsin-EDTA, Gibco, USA), Fetal Blood Serum (FBS, Gibco, USA, catalog number: 10099141C), type I collagenase (Worthington, USA), and CCK8 reagent (Cell Counting Kit-8, Dojindo, Japan).

[0074] 2 Methods

[0075] 2.1 Experiment on the promotion of hair follicle papilla proliferation by exosomes from adipose-derived stem cells

[0076] The 3rd to 5th generation rat hair follicle dermal papilla cells were inoculated into a 96-well plate (5000 cells / well), and 5 ADSC-Exo (prepared according to the method of Example 1) concentration gradients (0, 50, 100, 200, 400 μg / ml) and duplicate wells were set. Incubate overnight in a 5% carbon dioxide incubator at 37°C to allow it to adhere to the wall, discard the culture medium, wash 3 times with PBS, and then add the above-mentioned ADSC-Exo to incubate for 0, 24, 48, and 72 hours. Before detection, CCK8 reagent was added to DMEM basal culture medium at a ratio of 1:10, mixed and added to each well, incubated at 37°C for 1 hour, and the absorbance of OD450nm at 0h, 24h, 48h, and 72h of ADSC-Exo was detected with an enzyme marker to observe the proliferation of hair papilla cells.

[0077] 2.2 Animal experiments

[0078] Seven-week-old C57BL / 6 male mice were selected and anesthetized with 1% sodium pentobarbital intraperitoneally. The hair of a square area of ​​about 2*2cm on the back of the mice was shaved with a shaver and divided into two groups: PBS group and Exo (i.e. ADSC-Exo) group. The dosage of exosomes in the Exo group was 400μg / mouse subcutaneously injected into the hair removal area, and the PBS group was given an equal volume of PBS. The difference in hair growth between the two groups of mice was observed and recorded within 21 days after treatment.

[0079] 2. Results

[0080] 1 ADSC-Exo promotes the proliferation of hair follicle dermal papilla cells

[0081] The hair follicle papilla cell proliferation experiment showed that ADSC-Exo treatment can significantly promote cell proliferation ( Figure 5 ).

[0082] 2 ADSCs-Exo promotes hair growth in mice

[0083] The results of in vivo experiments in mice showed that hair growth in mice treated with ADSC-Exo was better than that in the PBS group (control group), and the number of hair follicles and dermal thickness were greater than those in the control group ( Figure 6 ). This indicates that the ADSCs-Exo prepared by this method has a hair-growth effect.

[0084] Example 3

[0085] 1. Materials and Methods

[0086] 1 Materials

[0087] 1.1 Animal sources

[0088] The experiments used SPF-grade C57BL / 6 male mice and SPF-grade SD male rats, which were purchased from Shanghai Slake Laboratory Animal Co., Ltd. and kept in the Experimental Animal Center of Tongji University. The experimental research ethics were approved by the Experimental Animal Center of Tongji University.

[0089] 1.2 Main experimental reagents / consumables

[0090] DMEM culture medium (Gibco, USA, catalog number: C11995500BT), phosphate buffer solution (PBS, Gibco, USA), 0.05% trypsin solution (Trypsin-EDTA, Gibco, USA), Fetal Blood Serum (FBS, Gibco, USA, catalog number: 10099141C), and type I collagenase (Worthington, USA). Ultra-15 Centrifugal Filter Unit (Millipore, USA, Catalog No. UFC910008), ExoQuick-TC (Catalog No. EXOTC10A-1, SBI Biosciences, SBI, USA), CCK8 reagent (Cell Counting Kit-8, Dojindo, Japan).

[0091] 2 Methods

[0092] 2.1 Exosome extraction method

[0093] 2.1.1 Preparation of Adipose-derived Stem Cells

[0094] (1) Adipose tissue was obtained from SPF-grade, 7-day-old C57BL / 6 mice. Bilateral inguinal fat was obtained under a sterile environment, and the blood vessels and fibrous components visible to the naked eye were removed.

[0095] (2) The adipose tissue specimens were rinsed repeatedly several times with a sterile PBS rinse solution containing 1% antibiotics (penicillin / streptomycin) to remove bloody fluid and fascia components and obtain pure adipose tissue.

[0096] (3) Use tissue scissors to cut the adipose tissue into chyme, then add 0.1% type I collagenase and digest at 37°C for 30 min to ensure uniform digestion of the tissue.

[0097] (4) Centrifuge at 2000 g for 10 min, discard the supernatant, add DMEM medium containing 10% fetal bovine serum, and place in a 37°C, 5% CO2 incubator for adherent culture.

[0098] (5) After 24 hours, change the medium and wash away the non-adherent cells and other impurities with PBS. Then add fresh culture medium. Change the medium every three days. When the cell density reaches 80%, use 0.05% trypsin for digestion and subculture.

[0099] 2.1.2 Preparation of Adipose-derived Stem Cell Exosomes

[0100] 2.1.2.1 Extraction method 1

[0101] (1) Adipose-derived stem cells of the third generation in the logarithmic growth phase were cultured in 10% exosome-free FBS DMEM in a 37° C., 5% CO 2 incubator for 48 hours, and the supernatant culture medium was collected.

[0102] (2) Centrifuge the supernatant at 3000 g for 15 min to remove cells and cell debris. Transfer the supernatant to a 100 kDa molecular weight ultrafiltration tube and centrifuge again at 1500 g for 30 min to concentrate the supernatant and collect the concentrate.

[0103] (3) Add ExoQuick-TC (SBI) reagent to the concentrate at a volume ratio of 1:5, mix gently, and incubate at 4°C for 12 h.

[0104] (4) Centrifuge the mixture at 4°C, 1500g for 30 min. Carefully discard the supernatant, add PBS and continue centrifugation at 1500g for 5 min, then remove the residual liquid. Add an appropriate amount of PBS or sterile water for injection, and gently blow the precipitate to obtain a resuspended solution.

[0105] 2.1.2.2 Extraction method 2

[0106] (1) The third generation of adipose-derived stem cells growing logarithmically were cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%. The original culture medium was aspirated, and the cells were washed three times with PBS. The cells were replaced with 10% exosome-free FBS DMEM culture medium and continued to be cultured in a 37°C, 5% CO2 incubator for 48 hours, and the supernatant culture medium was collected.

[0107] (2) Centrifuge the supernatant at 3000 g for 15 min to remove cells and cell debris. Transfer the supernatant to a 100 kDa molecular weight ultrafiltration tube and centrifuge again at 1500 g for 30 min to concentrate the supernatant and collect the concentrate.

[0108] (3) Add ExoQuick-TC (SBI) reagent to the concentrate at a volume ratio of 1:5, gently shake to mix, and place at 4°C for 12 h.

[0109] (4) Centrifuge the mixture at 4°C, 1500g for 30 min. Carefully discard the supernatant, add PBS and continue centrifugation at 1500g for 5 min, then remove the residual liquid. Add an appropriate amount of PBS or sterile water for injection, and gently blow the precipitate to obtain a resuspended solution.

[0110] 2.1.2.3 Extraction method 3

[0111] (1) The third generation of adipose-derived stem cells growing logarithmically were cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%. The original culture medium was aspirated, and the cells were washed three times with PBS. The cells were replaced with 10% exosome-free FBS DMEM culture medium and continued to be cultured in a 37°C, 5% CO2 incubator for 48 hours, and the supernatant culture medium was collected.

[0112] (2) Centrifuge the supernatant at 3000 g for 15 min to remove cells and cell debris. Transfer the supernatant to a 100 kDa molecular weight ultrafiltration tube and centrifuge again at 1000 g for 30 min to concentrate the supernatant and collect the concentrate.

[0113] (3) Add ExoQuick-TC (SBI) reagent to the concentrate at a volume ratio of 1:5, gently shake to mix, and place at 4°C for 12 h.

[0114] (4) Centrifuge the mixture at 4°C, 1500g for 30 min. Carefully discard the supernatant, add PBS and continue centrifugation at 1500g for 5 min, then remove the residual liquid. Add an appropriate amount of PBS or sterile water for injection, and gently blow the precipitate to obtain a resuspended solution.

[0115] 2.1.2.4 Extraction method 4

[0116] (1) The third generation of adipose-derived stem cells growing logarithmically were cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%. The original culture medium was aspirated, and the cells were washed three times with PBS. The cells were replaced with 10% exosome-free FBS DMEM culture medium and continued to be cultured in a 37°C, 5% CO2 incubator for 48 hours, and the supernatant culture medium was collected.

[0117] (2) Centrifuge the supernatant at 3000 g for 15 min to remove cells and cell debris. Transfer the supernatant to a 100 kDa molecular weight ultrafiltration tube and centrifuge again at 2000 g for 30 min to concentrate the supernatant and collect the concentrate.

[0118] (3) Add ExoQuick-TC (SBI) reagent to the concentrate at a volume ratio of 5:1, gently shake to mix, and incubate at 4°C for 12 h.

[0119] (4) Centrifuge the mixture at 4°C, 1500g for 30 min. Carefully discard the supernatant, add PBS and continue centrifugation at 1500g for 5 min, then remove the residual liquid. Add an appropriate amount of PBS or sterile water for injection, and gently blow the precipitate to obtain a resuspended solution.

[0120] 2.1.2.5 Extraction method 5

[0121] (1) The third generation of adipose-derived stem cells growing logarithmically were cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%. The original culture medium was aspirated, and the cells were washed three times with PBS. The cells were replaced with 10% exosome-free FBS DMEM culture medium and continued to be cultured in a 37°C, 5% CO2 incubator for 48 hours, and the supernatant culture medium was collected.

[0122] (2) Centrifuge the supernatant at 3000 g for 15 min to remove cells and cell debris. Transfer the supernatant to a 100 kDa molecular weight ultrafiltration tube and centrifuge again at 3000 g for 20 min to concentrate the supernatant and collect the concentrate.

[0123] (3) Add ExoQuick-TC (SBI) reagent to the concentrate at a volume ratio of 5:1, gently shake to mix, and incubate at 4°C for 12 h.

[0124] (4) Centrifuge the mixture at 4°C, 1500g for 30 min. Carefully discard the supernatant, add PBS and continue centrifugation at 1500g for 5 min, then remove the residual liquid. Add an appropriate amount of PBS or sterile water for injection, and gently blow the precipitate to obtain a resuspended solution.

[0125] 2.2.3 Observation of exosome morphology

[0126] The morphology of adipose stem cell exosomes was observed by transmission electron microscopy (TEM).

[0127] 2.2.4 Exosome production determination

[0128] The exosome concentration was detected using a BCA protein assay kit. Three samples were measured in each group and the experiment was repeated three times.

[0129] 2.2.5 ADSC-Exo promotes the proliferation of hair follicle dermal papilla cells

[0130] The 3rd-5th generation rat hair follicle dermal papilla cells were inoculated into a 96-well plate (5000 cells / well) and incubated overnight at 37°C in a 5% carbon dioxide incubator to adhere to the wall. The culture medium was discarded, and the cells were washed 3 times with PBS. ADSC-Exo was added to co-incubate with the dermal papilla cells. The maximum final concentration was 400μg / ml, and 3-5 replicate wells were set. CCK8 reagent was added to DMEM basal culture medium at a ratio of 1:10, mixed and added to each well, incubated at 37°C for 1h, and the absorbance at OD450nm was detected with an enzyme marker, which was counted as the 0h value. After that, ADSC-Exo was incubated for 24h, 48h, and 72h to observe the proliferation of dermal papilla cells. The experiment was repeated three times.

[0131] 2. Results

[0132] 1 Exosome morphological observation

[0133] Observation under an electron microscope showed that the exosomes obtained by the above extraction methods all showed the typical morphology of exosomes, which were round or oval vesicle-like structures.

[0134] 2 Comparison of exosome production

[0135] After testing, the exosome yield of method 2 was the highest, and this extraction method was selected as the most preferred method for extracting exosomes. The exosome yield was ranked from large to small as follows: extraction method 2 > extraction method 1 > extraction method 3 > extraction method 4 > extraction method 5. Compared with extraction method 2, the exosome yields of extraction methods 1, 3, 4, and 5 were significantly lower than those of extraction method 2 (P<0.05), indicating that the centrifugal force in the exosome concentration step has a significant effect on the exosome extraction efficiency.

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A use of mouse adipose stem cell exosomes in the preparation of a hair growth drug, characterized in that: The mouse adipose stem cell exosomes are prepared according to the following method: (a) Obtained logarithmically grown mouse adipose-derived stem cells from passages 3 to 6, cultured with 10% FBS-containing DMEM culture medium until the cell density reached 70%-80%, aspirated the original culture medium, washed with PBS, replaced with 10% exosome-free FBS-containing DMEM culture medium, and continued to culture in a 37°C, 5% CO2 incubator for 45-50 hours, and collected the supernatant culture medium; (b) The supernatant culture medium was centrifuged at 3000 g for 15 min to remove cells and cell debris, and the supernatant was transferred to a 100 kDa molecular weight ultrafiltration tube and centrifuged again at 1500-2500 g for 30 min to collect the concentrate; (c) Add the exosome extraction reagent to the concentrate, gently invert to mix, and place at 4°C for at least 12 h; (d) centrifuging the mixture at 4°C, 1500g×30min, carefully discarding the supernatant, adding PBS and continuing centrifugation at 1500g×5min, and removing the residual liquid to obtain the mouse adipose stem cell exosomes. The mouse adipose stem cells were isolated from 5-10 day old C57BL / 6 mice.

2. The use according to claim 1, characterized in that: In step (a), the culture medium was changed to DMEM medium containing 10% exosome-free FBS and the culture medium was continued for 48 hours, and the supernatant culture medium was collected.

3. The use according to claim 1, characterized in that: After removing the original culture medium in step (a), wash 1-3 times with PBS.

4. The use according to claim 1, characterized in that: The centrifugation parameters after the supernatant in step (b) is transferred to the ultrafiltration tube are: 1500g, 30min.

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