Pharmaceutical composition containing exosome and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a pharmaceutical composition containing exosomes and application of the pharmaceutical composition. The medicine composition containing the exosome is prepared from the stem cell exosome, recombinant IGFBP7 protein and cucurbitacin B, the amino acid sequence of the recombinant IGFBP7 protein is as shown in SEQ ID NO: 1. A test result shows that the umbilical cord mesenchymal stem cell exosome, the cucurbitacine B and the recombinant IGFBP7 protein are matched for use, so that hair follicle stem cells can be stimulated, the initial hair growth stage of hair follicles is prolonged, and the alopecia condition of a patient is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a pharmaceutical composition containing exosomes and its application. Background Art

[0002] Currently, for hair loss problems, there are mainly two treatment methods: drugs and hair transplantation. Among them, Rogaine, an FDA-approved hair loss drug, as the only proven effective hair growth drug on the market, its efficacy is limited to the short term and cannot fundamentally improve the scalp microenvironment. Once the drug is stopped, the hair growth condition will quickly return to the pre-treatment state, and long-term use may cause side effects such as hormonal imbalance. On the other hand, hair transplantation, as an invasive and expensive treatment method, although feasible, does not change the follicular microenvironment. Therefore, the newly transplanted hair is prone to fall out again, and the problem has not been fundamentally solved. It should be noted that stem cell exosome products are currently mostly used in tissue repair and beauty fields, and their application in hair loss prevention and treatment is still rare, especially in the treatment of common androgenetic alopecia.

[0003] It is known that chronic inflammation around hair follicles is one of the important factors leading to androgenetic alopecia. Inflammation can cause apoptosis of follicular cells, further accelerating the degeneration and atrophy of hair follicles, thus affecting hair growth. For the inflammatory factors in androgenetic alopecia, anti-inflammatory treatment may be an effective treatment method. By using anti-inflammatory drugs or physical therapy and other means, the inflammatory reaction around hair follicles can be reduced, thereby delaying the process of hair loss. Traditional Chinese medicine extracts have high activity and low toxicity and side effects, and have the potential to treat hair loss. Cucurbitacin B has strong anti-inflammatory activity, but the research on its treatment of androgenetic alopecia is still blank.

[0004] In addition, it has been reported that the levels of IGFBP7 in the serum and scalp follicles of AGA patients are lower than those of healthy controls. Subcutaneous injection of IGFBP7 delayed the onset of the telogen phase in DHT-induced AGA mice and prolonged the anagen phase of scalp follicles. It has great potential for the treatment of AGA. However, the existing recombinant IGFBP7 protein has a very complex structure, and it is difficult to artificially express and mass-produce, with high costs and poor protein stability. Therefore, developing a recombinant IGFBP7 protein with a smaller molecular weight and higher stability has become a key problem to be solved urgently. Summary of the Invention

[0005] The first object of the present invention is to provide a pharmaceutical composition containing exosomes, which can stimulate hair follicle stem cells, prolong the anagen phase of hair follicles, and thus effectively improve the hair loss situation of AGA patients.

[0006] The second object of the present invention is to provide an application of a pharmaceutical composition containing exosomes, which has broad application prospects.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A pharmaceutical composition containing exosomes, the pharmaceutical composition comprising stem cell exosomes, recombinant IGFBP7 protein, and bruceine B; the amino acid sequence of the recombinant IGFBP7 protein is as shown in SEQ ID NO:1.

[0009] The sequence of SEQ ID NO:1 is as follows:

[0010] PGVSGVCVCKSGYPVCGSDGLAYPSGCQLRAASQRAESRGEGAITQVHKGTCEQPHIV

[0011] TPPKDIWNVTGAQVYLSCEVCGIPTPVLIWNKVKRGHYGVQRTELLPGDRDNLHGQTRGG

[0012] PEKHEVTGWVLVSPLSKEDAGEYECHASNSAGQASASAKHT

[0013] Furthermore, the nucleotide sequence of the recombinant IGFBP7 protein is as shown in SEQ ID NO:2.

[0014] The sequence of SEQ ID NO:2 is as follows:

[0015] CCGGGCGTATCTGGTGTATGTGTCTGCAAGTCTGGCTACCCGGTGTGCGGCAGCGA

[0016] CGGCCTGGCCTACCCCAGCGGCTGCCAGCTGAGAGCCGCCAGCCAGAGAGCCGAGAGC

[0017] AGAGGCGAGGGCGCCATCACCCAGGTGCACAAGGGCACCTGCGAGCAGCCCCACATCG

[0018] TGACCCCCCCCAAGGACATCTGGAACGTGACCGGCGCCCAGGTGTACCTGAGCTGCGA

[0019] GGTGTGCGGCATCCCCACCCCCGTGCTGATCTGGAACAAGGTGAAGAGAGGCCACTAC

[0020] GGCGTGCAGAGAACCGAGCTGCTGCCCGGCGACAGAGACAACCTGCACGGCCAGACC

[0021] AGAGGCGGCCCCGAGAAGCACGAGGTGACCGGCTGGGTGCTGGTGAGCCCCCTGAGC

[0022] AAGGAGGACGCCGGCGAGTACGAGTGCCACGCCAGCAACAGCGCCGGCCAGGCCAGC

[0023] GCCAGCGCCAAGCACACC

[0024] Furthermore, the mass ratio of stem cell exosomes, recombinant IGFBP7 protein, and bruceine B in the pharmaceutical composition is 1:(1 - 2):1.

[0025] Furthermore, the preparation method of the recombinant IGFBP7 protein is as follows:

[0026] (1) Connect the nucleotide sequence encoding the recombinant IGFBP7 protein with a vector to obtain a recombinant plasmid;

[0027] (2) Transform the recombinant plasmid in step (1) into bacteria to construct a recombinant bacterium;

[0028] (3) Express the recombinant bacterium constructed in step (2), collect the cell pellet, break it to obtain a supernatant, and purify to obtain the recombinant IGFBP7 protein.

[0029] Furthermore, the process of the expression in step (3) is as follows: inoculate the recombinant bacterium into an LB medium containing kanamycin and shake overnight, and add IPTG to induce the expression of the recombinant IGFBP7 protein; the induction expression concentration of IPTG is 0.3 - 0.5 mmol / L, the induction expression time of IPTG is 5 - 10 h; the concentration of kanamycin is 60 - 90 μg / mL.

[0030] Furthermore, the stem cell exosomes are umbilical cord mesenchymal stem cell exosomes.

[0031] Furthermore, the acquisition method of the umbilical cord mesenchymal stem cell exosomes is as follows:

[0032] Inoculate passage P2 - P5 umbilical cord mesenchymal stem cells into a medium for culture at a density of (1 - 5)×10 4 cells / mL, collect the cell culture supernatant, separate the cell culture supernatant by ultracentrifugation, collect the precipitate, and freeze - dry to obtain umbilical cord mesenchymal stem cell exosomes.

[0033] Furthermore, the culture medium consists of DMEM / F12 basal medium and the following components added thereto: 8-10% FBS, 90-100 ng / mL IL-2, 30-50 ng / mL IL-21, 1-5 ng / mL EGF; the culture time is 2-3 days, and the culture conditions are 37°C and 5% CO2.

[0034] Use of the above drug composition containing exosomes in the preparation of a product for treating hair loss.

[0035] Furthermore, based on the total mass of the product, the addition amount of the drug composition is 2-8%.

[0036] Compared with the prior art, the beneficial effects of the present invention mainly lie in:

[0037] The present invention provides a drug composition, which is composed of umbilical cord mesenchymal stem cell exosomes, recombinant IGFBP7 protein, and bruceine B. The recombinant IGFBP7 protein is obtained by intercepting and designing effective fragments of the amino acid sequence of the full-length human IGFBP7 protein. The recombinant IGFBP7 protein has a smaller molecular weight, better stability, and stronger activity. The test results show that the combination of umbilical cord mesenchymal stem cell exosomes, bruceine B, and recombinant IGFBP7 protein can stimulate hair follicle stem cells and prolong the anagen phase of hair follicles, thereby effectively improving the hair loss condition of AGA patients. Specifically, umbilical cord mesenchymal stem cell exosomes can promote hair follicle repair and regeneration and shorten the telogen phase of hair follicles; bruceine B can reduce inflammatory reactions and improve local microcirculation, thereby accelerating the entry of telogen hair follicles into the anagen phase. The recombinant IGFBP7 protein of the present invention can better promote the in vitro migration of human outer root sheath cells and prolong the anagen phase of hair follicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a morphological diagram of passage 3 umbilical cord mesenchymal stem cells;

[0039] Figure 2 is a result diagram of SDS-PAGE electrophoresis for identifying recombinant IGFBP7 protein, Figure 2 in which "M" represents a protein marker, and "1" represents lane 1, namely the purified recombinant protein IGFBP7. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. Specific conditions not indicated in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0041] The product number of the commercially available recombinant IGFBP7 protein of the present invention is abs04667.

[0042] Example

[0043] Example 1

[0044] In Example 1, umbilical cord mesenchymal stem cell exosomes were obtained, and the specific method was as follows:

[0045] The umbilical cord tissue was washed with PBS buffer, cut into 1 mm 3 tissue pieces with surgical scissors and spread them on a 10 cm culture dish (serum-free DMEM / F12 medium containing 10% fetal bovine serum), and cultured in a 37 °C, 5% CO2 cell culture incubator until the cell confluence reached 80%. After digestion with Tryspin-EDTA enzyme, primary cultured umbilical cord mesenchymal stem cells were obtained; at a density of 3×10 4 cells / mL, the primary cultured umbilical cord mesenchymal stem cells were inoculated into the medium, and the medium consisted of DMEM / F12 basal medium and the following components added to the medium: 10% FBS, 100 ng / mL IL-2, 50 ng / mL IL-21, 5 ng / mL EGF. When the cell confluence reached 90%, the cells were digested with 0.25% EDTA trypsin solution, and passaged in a 37 °C, 5% CO2 cell culture incubator. The passage ratio was 1:2, and continuous passage was carried out 3 times to obtain P3-generation umbilical cord mesenchymal stem cells. The morphological diagram of P3-generation umbilical cord mesenchymal stem cells is as Figure 1 shown.

[0046] At a density of 3×10 4At a density of cells / mL, passage 3 umbilical cord mesenchymal stem cells were seeded into the culture medium. The culture medium consisted of DMEM / F12 basal medium and the following components added to the medium: 10% FBS, 100 ng / mL IL-2, 50 ng / mL IL-21, 5 ng / mL EGF. The cells were cultured in a 37°C, 5% CO2 cell incubator for 2 days, and the cell culture supernatant was collected. The cell supernatant was centrifuged at low speed at 400 g for 8 min, then the collected supernatant was centrifuged at medium speed at 4500 g for 6 min, and then the collected supernatant was ultracentrifuged at 85000 g for 30 min. The precipitate was collected, resuspended and then ultracentrifuged at 85000 g for 30 min. After lyophilization, umbilical cord mesenchymal stem cell exosomes were obtained.

[0047] Example 2

[0048] The recombinant IGFBP7 protein was obtained in Example 2. The specific preparation process was as follows:

[0049] (1) Based on the amino acid sequence of the human full-length IGFBP7 protein, functional fragments were truncated and designed. The amino acid sequence of the recombinant IGFBP7 protein is shown in SEQ ID NO:1. According to the amino acid sequence of the recombinant IGFBP7 protein, the gene sequence was designed in reverse, and the corresponding base sequence was codon-optimized according to the codon preference of Escherichia coli for heterologous protein expression. The nucleotide sequence of the recombinant IGFBP7 protein is shown in SEQ ID NO:2. The nucleotide sequence of the recombinant IGFBP7 protein was ligated to the pET28a vector, and after identification and sequencing, the recombinant plasmid IGFBP7-pET28a was obtained.

[0050] The sequence of SEQ ID NO:1 is as follows:

[0051] PGVSGVCVCKSGYPVCGSDGLAYPSGCQLRAASQRAESRGEGAITQVHKGTCEQPHIV

[0052] TPPKDIWNVTGAQVYLSCEVCGIPTPVLIWNKVKRGHYGVQRTELLPGDRDNLHGQTRGG

[0053] PEKHEVTGWVLVSPLSKEDAGEYECHASNSAGQASASAKHT

[0054] The sequence of SEQ ID NO:2 is as follows:

[0055] CCGGGCGTATCTGGTGTATGTGTCTGCAAGTCTGGCTACCCGGTGTGCGGCAGCGACGGCCTGGCCTACCCCAGCGGCTGCCAGCTGAGAGCCGCCAGCCAGAGAGCCGAGAGCAGAGGCGAGGGCGCCATCACCCAGGTGCACAAGGGCACCTGCGAGCAGCCCCACATCGTGACCCCCCCCAAGGACATCTGGAACGTGACCGGCGCCCAGGTGTACCTGAGCTGCGAGGTGTGCGGCATCCCCACCCCCGTGCTGATCTGGAACAAGGTGAAGAGAGGCCACTACGGCGTGCAGAGAACCGAGCTGCTGCCCGGCGACAGAGACAACCTGCACGGCCAGACCAGAGGCGGCCCCGAGAAGCACGAGGTGACCGGCTGGGTGCTGGTGAGCCCCCTGAGCAAGGAGGACGCCGGCGAGTACGAGTGCCACGCCAGCAACAGCGCCGGCCAGGCCAGCGCCAGCGCCAAGCACACC

[0056] (2) Transform the recombinant plasmid obtained in step (1) into the engineered bacterium E. coli BL21(DE3), evenly coat the recombinant bacterium on an LB agar plate containing 70 μg / mL kanamycin, and incubate overnight at 37 °C to obtain the recombinant bacterium IGFBP7-pET28a / BL21.

[0057] (3) Pick a positive single colony of the recombinant bacterium IGFBP7-pET28a / BL21 obtained in step (2) and inoculate it into 4 mL of LB medium containing 50 μg / mL kanamycin, and culture it overnight with shaking at 37 °C and 230 rpm. Then inoculate the colonies into LB medium containing 70 μg / mL kanamycin according to 1% of the total volume of the medium, and culture it with shaking at 37 °C and 220 rpm for 3 h until OD 600 reaches 0.6, add IPTG with a final concentration of 0.4 mM, induce for 8 h at 16 °C and 180 rpm, then centrifuge to collect the bacterial cells, obtain the crude protein through separation and affinity chromatography, and then obtain the purified recombinant IGFBP7 protein through ion exchange chromatography. Use SDS-PAGE electrophoresis to identify the purity of the recombinant protein, and the results are as Figure 2 shown.

[0058] Figure 2 This is the result diagram of identifying the recombinant IGFBP7 protein by SDS-PAGE electrophoresis. From Figure 2It can be seen that the recombinant IGFBP7 protein has a high purity. The estimated molecular weight of the recombinant protein is 17 kDa, and the band position is consistent with the expected size.

[0059] Example 3

[0060] Example 3 provides a drug composition containing exosomes. This composition consists of umbilical cord mesenchymal stem cell exosomes prepared in Example 1, recombinant IGFBP7 protein prepared in Example 2, and bruceine B, wherein the mass ratio of umbilical cord mesenchymal stem cell exosomes, recombinant IGFBP7 protein, and bruceine B is 1:1:1.

[0061] Example 4

[0062] Example 4 provides a drug composition containing exosomes. This composition consists of umbilical cord mesenchymal stem cell exosomes prepared in Example 1, recombinant IGFBP7 protein prepared in Example 2, and bruceine B, wherein the mass ratio of umbilical cord mesenchymal stem cell exosomes, recombinant IGFBP7 protein, and bruceine B is 1:1.5:1.

[0063] Example 5

[0064] Example 5 provides a drug composition containing exosomes. This composition consists of umbilical cord mesenchymal stem cell exosomes prepared in Example 1, recombinant IGFBP7 protein prepared in Example 2, and bruceine B, wherein the mass ratio of umbilical cord mesenchymal stem cell exosomes, recombinant IGFBP7 protein, and bruceine B is 1:2:1.

[0065] Comparative Example

[0066] Comparative Example 1

[0067] This comparative example provides a drug composition, which is different from that of Example 4 in that: the drug composition of this comparative example consists of umbilical cord mesenchymal stem cell exosomes prepared in Example 1 and recombinant IGFBP7 protein prepared in Example 2, wherein the mass ratio of umbilical cord mesenchymal stem cell exosomes to recombinant IGFBP7 protein is 1:1.5.

[0068] Comparative Example 2

[0069] This comparative example provides a drug, which is different from that of Example 4 in that: the drug of this comparative example is umbilical cord mesenchymal stem cell exosomes prepared in Example 1, and the dosage of umbilical cord mesenchymal stem cell exosomes is the same as the total dosage of umbilical cord mesenchymal stem cell exosomes, recombinant IGFBP7 protein, and bruceine B in Example 4.

[0070] Comparative Example 3

[0071] This comparative example provides a drug, which is different from that of Example 4 in that: the drug in this comparative example is the recombinant IGFBP7 protein prepared in Example 2, and the dosage of the recombinant IGFBP7 protein is the same as the total dosage of umbilical cord mesenchymal stem cell exosomes, recombinant IGFBP7 protein, and dapsone in Example 4.

[0072] Comparative Example 4

[0073] This comparative example provides a pharmaceutical composition, which is different from that of Example 4 in that: the pharmaceutical composition in this comparative example is composed of umbilical cord mesenchymal stem cell exosomes prepared in Example 1, commercially available recombinant IGFBP7 protein, and dapsone, and the mass ratio of umbilical cord mesenchymal stem cell exosomes, commercially available recombinant IGFBP7 protein, and dapsone is 1:1.5:1.

[0074] The performance of the pharmaceutical composition of the present invention is described below.

[0075] The effects of the drugs obtained in Examples 3-5 and Comparative Examples 1-4 were detected as follows:

[0076] Experimental subjects: A total of 100 6-week-old SPF-grade male C57BL / 6 mice.

[0077] Feeding environment: The temperature is (23±2)°C, the relative humidity is 40%-60%, and the light and darkness are each 12 h.

[0078] AGA model establishment: All mice were adaptively fed for one week and anesthetized with ether. Rosin and paraffin were heated and melted (1:1), and after cooling, they were applied to a 2 cm×2 cm area on the back of the mice. After solidification, they were peeled off to confirm that the hair of the mice was in the telogen phase (the skin was pink). On the second day, the mice were randomly divided into a blank group, a model group, Examples 3-5, and Comparative Examples 1-4 groups, with 10 mice in each group.

[0079] Drug administration: Except for the blank group, 200 μL of 0.05% testosterone (prepared with 75% ethanol) was applied to the depilated area of the remaining groups once a day for 28 consecutive days. After 30 min of testosterone treatment, 200 μL of the drug was given in the same method. The blank group and the model group were given 75% ethanol, and the remaining groups were given the drugs of Examples 3-5 and Comparative Examples 1-4 at 40 mg / mL (both prepared with 75% ethanol).

[0080] Before applying the drug every day, the back skin of each group of mice was observed, and the time when the back skin changed color (began to turn gray) was recorded; after 28 days of treatment, the mice in each group were decapitated and sacrificed, and the hair in the upper left, lower left, middle, upper right, and lower right regions of the depilated area of the mice was plucked and measured with a vernier caliper. The hair in the depilated area of the mice was shaved off with a razor and weighed. The final data was the average value of the detection data of each group, and the results are shown in Table 1.

[0081] Table 1

[0082]

[0083]

[0084] As can be seen from the above, compared with the blank group, the skin discoloration time and hair growth time of the mice in the model group were significantly delayed, and the hair length was shorter and the hair quality was lighter.

[0085] Compared with the model group, after using the drugs obtained in Examples 3-5, the skin discoloration time and hair growth time of the mice were advanced by 2 days, and the hair length and hair quality were significantly increased. The above results indicate that the drugs obtained in Examples 3-5 can alleviate the inhibitory effect of testosterone on hair growth in C57BL / 6 mice.

[0086] After using the drugs obtained in Comparative Examples 1-3, the effects were inferior to those in Examples 3-5. Analysis showed that colchicine B was not added in Comparative Example 1; recombinant IGFBP7 protein and colchicine B were not added in Comparative Example 2, but the dosage of umbilical cord mesenchymal stem cell exosomes was increased; umbilical cord mesenchymal stem cell exosomes and colchicine B were not added in Comparative Example 3, but the dosage of recombinant IGFBP7 protein was increased. Therefore, the above results indicate that the combined use of umbilical cord mesenchymal stem cell exosomes, colchicine B, and recombinant IGFBP7 protein can stimulate hair follicle stem cells and extend the initial stage of hair growth in hair follicles, thereby effectively improving the hair loss situation of AGA patients. Analyzing the reasons, it may be that umbilical cord mesenchymal stem cell exosomes can promote hair follicle repair and regeneration and shorten the resting period of hair follicles; colchicine B can reduce inflammatory reactions and improve local microcirculation, thereby accelerating the entry of resting hair follicles into the growth phase. The recombinant IGFBP7 protein of the present invention can better promote the in vitro migration of human outer root sheath cells and extend the initial stage of hair growth in hair follicles.

[0087] Compared with Examples 3-5, after using the drug obtained in Comparative Example 4, the back skin and hair growth of the mice were slightly worse. This may be because in Comparative Example 4, a commercially available recombinant IGFBP7 protein was used instead of the recombinant IGFBP7 protein prepared in the present invention. Compared with the commercially available recombinant IGFBP7 protein, the recombinant IGFBP7 protein obtained in the present invention has a smaller molecular weight, better activity and stability, and can thus more effectively alleviate the inhibitory effect of testosterone on hair growth in C57BL / 6 mice.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. The basic principles and main features of the present invention have been described with specific implementation schemes above. On the basis of the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of protection required by the present invention.

Claims

1. A pharmaceutical composition containing exosomes, characterized in that: The pharmaceutical composition comprises stem cell exosomes, recombinant IGFBP7 protein and sciuronan B; the amino acid sequence of the recombinant IGFBP7 protein is shown in SEQ ID NO:

1.

2. The pharmaceutical composition containing exosomes according to claim 1, characterized in that: The nucleotide sequence of the recombinant IGFBP7 protein is shown in SEQ ID NO:

2.

3. The pharmaceutical composition containing exosomes according to claim 1, characterized in that: The mass ratio of stem cell exosomes, recombinant IGFBP7 protein and cholinesterol B in the pharmaceutical composition is 1:(1-2):

1.

4. The pharmaceutical composition containing exosomes according to claim 1, characterized in that The preparation method of the recombinant IGFBP7 protein is as follows: (1) connecting the nucleotide sequence encoding the recombinant IGFBP7 protein to a vector to obtain a recombinant plasmid; (2) transforming the recombinant plasmid of step (1) into bacteria to construct recombinant bacteria; (3) Expressing the recombinant bacteria constructed in step (2), collecting bacterial precipitates, crushing them to obtain supernatant, and purifying them to obtain recombinant IGFBP7 protein.

5. The pharmaceutical composition containing exosomes according to claim 4, characterized in that: The expression process in step (3) is as follows: the recombinant bacteria are inoculated into LB medium containing kanamycin and shaken overnight, and IPTG is added to induce the expression of the recombinant IGFBP7 protein; the induction expression concentration of the IPTG is 0.3-0.5 mmol / L, and the induction expression time of the IPTG is 5-10 h; the concentration of the kanamycin is 60-90 μg / mL.

6. The pharmaceutical composition containing exosomes according to claim 1, characterized in that: The stem cell exosomes are umbilical cord mesenchymal stem cell exosomes.

7. The pharmaceutical composition containing exosomes according to claim 4, characterized in that: The method for obtaining the umbilical cord mesenchymal stem cell exosomes is: According to (1-5)×10 4 The P2-P5 umbilical cord mesenchymal stem cells were inoculated into the culture medium at a density of 100 cells / mL, the cell culture supernatant was collected, the cell culture supernatant was separated by ultracentrifugation, the precipitate was collected, and the umbilical cord mesenchymal stem cell exosomes were obtained by freeze-drying.

8. The pharmaceutical composition containing exosomes according to claim 5, characterized in that: The culture medium consists of DMEM / F12 basic culture medium and the following components added to the culture medium: 8-10% FBS, 90-100ng / mL IL-2, 30-50ng / mL IL-21, 1-5ng / mL EGF; the culture time is 2-3 days, and the culture conditions are 37°C and 5% CO2.

9. Use of the exosome-containing pharmaceutical composition according to any one of claims 1 to 8 in the preparation of a product for treating hair loss.

10. Use of the exosome-containing pharmaceutical composition according to claim 9 in preparing a product for treating hair loss, characterized in that: Based on the total mass of the product, the added amount of the pharmaceutical composition is 2-8%.

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