CN116602992A

CN116602992A - Use of brown adipocyte-derived exosomes in the treatment of arthritis-related disorders

Info

Publication number: CN116602992A
Application number: CN202310174941.6A
Authority: CN
Inventors: 金万洙; 叶荣财; 黄园园; 蒋瑞
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2022-04-20
Filing date: 2023-02-28
Publication date: 2023-08-18
Also published as: CN114517187A

Abstract

The invention discloses application of brown adipocyte-derived exosomes in treating arthritis-related diseases, and belongs to the technical field of molecular biology. The preparation method of the exosome provided by the invention comprises the steps of constructing a humanized skin fibroblast which coexpresses PPARgamma and CEBP beta to obtain a brown fat precursor cell; inducing differentiation culture is carried out on brown fat precursor cells by using a brown fat cell inducing differentiation culture medium to obtain brown fat cells; and (3) secreting the brown adipocytes into exosomes to obtain the exosomes derived from the brown adipocytes. The exosome provided by the invention can treat arthritis pain symptoms through an external application administration mode, so that the exosome can be used for preparing medicines for treating arthritis related diseases.

Description

Use of brown adipocyte-derived exosomes in the treatment of arthritis-related disorders

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to application of an exosome derived from brown adipocytes transdifferentiated by human skin fibroblasts (HEF cells) in treating arthritis-related diseases.

Background

The arthritic condition includes inflammation of one or more joints, and the main symptoms are joint pain, stiffness, limited movement, fever and redness of the skin of the joint, muscle weakness, muscular atrophy and the like, and the cause of the inflammation is not completely known, but is related to multiple factors including congenital genetic factors, acquired environmental factors, age, sex, obesity, joint injury, excessive use of the joint and the like.

According to the latest epidemiological studies, at least about 6 millions of people suffering from arthritis are present in China, and the number of patients is gradually increased. Arthritis affects not only the quality of life of the patient, but such a large disease base also places a great burden on society and national production. At present, certain toxic and side effects or traumatic injury defects exist in the treatment scheme aiming at arthritis. Thus, how to benefit arthritic patients in a non-invasive and safe manner is currently a focus of attention.

With the deep research of stem cells and the general application of bioengineering technology, more and more researchers explore the application of the technology in the field of arthritis treatment, and gradually demonstrate more optimistic application prospects. However, the existing stem cell therapy for arthritis is mainly to inject stem cells in situ at a patient, but the injection mode is invasive therapy, which inevitably has a certain risk of local hemorrhage, hematoma and the like, and especially when aseptic operation is improper, local infection can be caused. In addition, if the injection is not proper, local tissue embolism may be caused, and local tissue necrosis may be caused.

Disclosure of Invention

In view of the problems in the prior art, one aspect of the present invention provides the use of brown adipocyte-derived exosomes in the manufacture of a medicament for treating arthritis-related conditions; wherein:

the medicine is an external medicine;

the brown adipocyte-derived exosomes are prepared by a method comprising the steps of:

s1: constructing a humanized skin fibroblast co-expressing PPARgamma and CEBP beta to obtain a brown fat precursor cell;

s2: performing induction differentiation culture on the brown fat precursor cells obtained in the step S1 by using a brown fat cell induction differentiation culture medium to obtain brown fat cells;

s3: and (3) secreting the brown adipocytes obtained in the step (S2) into exosomes to obtain the exosomes derived from the brown adipocytes.

In some embodiments, the operation of constructing a human skin fibroblast co-expressing pparγ and CEBP β described in step S1 is:

s11: respectively constructing a lentiviral expression vector for expressing PPARgamma and CEBP beta to obtain a PPARgamma lentiviral expression vector and a CEBP beta lentiviral expression vector;

s12: respectively carrying out slow virus packaging on the PPARgamma slow virus expression vector and the CEBP beta slow virus expression vector which are constructed in the step S11 to obtain a slow virus for expressing PPARgamma and a slow virus for expressing CEBP beta;

s13: lentiviral infection of human skin fibroblasts was performed using the lentivirus expressing pparγ and the lentivirus expressing cebpβ obtained in step S12 to obtain the brown fat precursor cells.

In some embodiments, the coding sequences of PPARgamma and CEBP beta described in step S1 are set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively.

In some embodiments, the formulation of the brown adipocyte-induced differentiation medium of step S2 comprises: 97-99% by volume of high sugar DMEM, 1-3% by volume of fetal bovine serum, 32-34. Mu.M biotin, 0.4-0.6. Mu.M insulin, 16-18. Mu.M pantothenic acid, 0.09-0.11. Mu.M DEX, 1-3. Mu.M T3, 0.50-0.60mM IBMX and 0.02-0.04mM indomethacin.

In some embodiments, the time for inducing the differentiation culture in step S2 is 6 days or more.

In some embodiments, the conditions for inducing differentiation culture in step S2 are 5% co ₂ ，37±1℃。

In some embodiments, the step S3 of secreting exosomes from the brown adipocytes obtained in step S2 is performed using a serum-free basal medium.

In another aspect, the present invention provides a medicament for treating arthritis-related conditions, comprising as an active ingredient the brown adipocyte-derived exosomes mentioned above; the medicine is an external medicine.

In some embodiments, the dosage form of the medicament includes powders, solutions, tinctures, spirits, lotions, ointments, creams, pastes, oils, gels, films, wipes, drops, aerosols, wet packs, suppositories, patches and sprays.

In some embodiments, the arthritis-related conditions include gouty arthritis, cervical disc herniation, lumbar disc lesions, and tendinitis.

Based on the above technical scheme, the present invention can obtain a brown adipocyte capable of secreting exosomes by coexpression of pparγ and CEBP β by human skin fibroblasts at the differentiation terminal (having lost totipotent stem property), and the results of examples prove that exosomes secreted by the brown adipocyte can be used as an active ingredient of an external medicine for treating arthritis-related disorders, and have excellent effects of non-invasively improving pain symptoms of arthritis, can significantly relieve pain of patients, and have no or little side effects, so that the exosomes can be applied to the preparation of external medicine for treating and improving arthritis-related disorders including gouty arthritis, herniation of cervical disc, herniation of lumbar disc, pathology of lumbar disc, and tendinitis.

Drawings

FIG. 1 shows cell morphology of GFP-stably expressing HEF cells, monostatically expressing HEF cells of CEBP beta as cells after induction culture of HEF cells stably co-expressing PPARgamma and CEBP beta in brown adipocyte-induced differentiation medium.

FIG. 2 is a plot of average thickness of paw swelling over time for each group of mice measured on days 21 to 42 (0 w-3 w) in a collagen-induced mouse model of arthritis.

FIG. 3 shows the levels of inflammatory factor TNFa in serum from each group of mice collected on day 42 (3 w) in a collagen-induced mouse arthritis model animal.

Fig. 4 is an image of paraffin embedded sections at knee joints of collagen-induced mouse arthritis model animals.

Detailed Description

Aiming at a plurality of defects of the arthritis treatment modes in the prior art, the invention provides an external medicine for treating arthritis related diseases, which takes exosomes derived from brown adipocytes transdifferentiated by human skin fibroblasts as active ingredients.

The pathological changes of arthritis are mainly characterized by inflammation in joints, so that the current treatment method for arthritis mainly delivers medicines to the inflammation parts in joints (such as by injection and the like) so as to achieve the purpose of in-situ elimination of inflammation. The stratum corneum is an important barrier against external irritation, and it is well pointed out in pharmaceutics that substances with a molecular weight of more than 600Da are more difficult to pass through the stratum corneum. While exosomes are vesicles with a diameter of 40-100nm, which are generally considered to have a molecular weight of greater than 100KDa, there has been no report to date of using exosomes as external drugs for treating diseases under the stratum corneum (e.g. intra-articular) of the skin, and even though there has been a report of using exosomes for treating osteoarthritis, they are delivered to the site of intra-articular inflammation in a subject by injection. However, the present inventors have surprisingly found that when using exosomes derived from brown adipocytes transdifferentiated human skin fibroblasts as active ingredients of an external drug for treating arthritis, the results of the examples demonstrate that the external drug can treat arthritis-related disorders with good therapeutic effects, and that the external drug used is a non-invasive treatment modality, which can effectively avoid risks including local bleeding, hematoma, local infection, local tissue embolism, local tissue necrosis, etc. that may exist in invasive treatment modalities (e.g., injection) of the prior art. Based on the above, the invention provides application of exosomes derived from brown adipocytes transdifferentiated by human skin fibroblasts in preparing medicines (especially external medicines) for treating arthritis-related diseases, and provides an external medicine for treating arthritis-related diseases.

The invention is further illustrated below in conjunction with specific examples. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The methods used in the examples described below are conventional methods unless otherwise specified. The specific steps can be seen in: molecular cloning guidelines (Molecular Cloning: A Laboratory Manual) Sambrook, j., russell, david w., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).

The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples. The experimental materials used in the examples described below, unless otherwise specified, are all conventional biochemical reagents and are commercially available.

The sequences referred to in the examples below can all be synthesized using the prior art.

Example 1: modification of HEF cells (human skin fibroblasts) to precursor cells with the potential to differentiate into brown adipocytes

This example aims at engineering HEF cells into precursor cells having the potential to differentiate into brown adipocytes and identifying the characteristics of the precursor cells, and specifically comprises the following steps.

1.1 plasmid construction

The human PPARgamma cDNA sequence (the nucleotide sequence of which is shown as SEQ ID NO: 1) and the human CEBP beta cDNA sequence (the nucleotide sequence of which is shown as SEQ ID NO: 2) are cloned into a pCDH-EF1-MCS-T2A-Gfp vector (commercially available) and a pCDH-EF1-MCS-T2A-Puro vector (commercially available) respectively, and the PPARgamma virus expression vector and the CEBP beta virus expression vector are obtained respectively.

1.2 lentiviral packaging

HEK 293T cells were cultured (complete medium for cell culture was complete medium containing 10% FBS) and the cells were packed at a density of 8X 10 ⁵ Individual cells/cm ² The cells were evenly inoculated into 6cm dishes and ready for transfection when the cells had fused to 70-80%. DMEM serum-free medium and DMEM 10% fbs-free double antibody medium were prepared for use. Viral expression vector during packaging (obtained in step 1.1): psPAX2: the mass ratio of pMD2G was 5 μg:2 μg: 1. Mu.g as transfection mixture. Specifically, 16. Mu.l Lipofectamine2000 was added to the plasmid solution, and the mixture was homogenized and incubated at 37℃for 5 minutes. Meanwhile, the HEK 293T cell culture medium is discarded, and replaced by 2.5ml of DMEM culture medium containing 10% FBS and not containing double antibodies, and then a transfection mixture is added; after 12h, the DMEM complete culture medium is changed, after 48h, 1ml of each EP tube is supplemented, after 72h, the culture medium containing the virus is respectively transferred into a centrifuge tube, and the centrifugation is carried out at 3000r/min and 4 ℃ for 10min, so that the virus liquid is removed. The virus solutions were filtered with 0.45 μm filters, respectively, and stored in a-80℃refrigerator for use. The lentivirus producing PPARgamma and the lentivirus producing CEBP beta are obtained respectively.

1.3 HEF cell construction to stably transduce PPARgamma and CEBP beta

Recovering HEF cells stored in frozen state according to the inoculation density of 8×10 ⁵ Individual cells/cm ² Inoculated in 6cm dishes, 4 ml/dish. The complete medium for cell culture was 10% FBS-containing complete medium. At 5% CO ₂ Culturing in an incubator at 37 ℃. After the HEF cells are fused to 80-90%, the PPARgamma-producing lentivirus and CEBP beta-producing lentivirus obtained in step 1.2 are used to infectHEF cells are subjected to GFP flow screening and PURO pressure screening after 48 hours, and HEF cells which can stably co-express PPARgamma and CEBP beta in a co-high mode are obtained, namely precursor cells with the potential of differentiating into brown adipocytes. In this step, HEF cells stably expressing GFP, HEF cells monostable expressing CEBP beta and HEF cells monostable expressing PPARgamma were constructed simultaneously in the above manner.

1.4 characterization of precursor Stem cells with potential to differentiate into brown adipocytes

The GFP-stably expressing HEF cells, CEBP β -monostabilized and pparγ -monostabilized HEF cells constructed in step 1.3 and the pparγ -stably co-expressing and CEBP β -stably co-expressing HEF cells were inoculated into 12-well plates, respectively, at a cell density of 100% (under a microscope, the cells were in contact with each other). After 24 hours, the cells were cultured by replacing them with brown adipocyte-induced differentiation medium (shown in Table 1 below) for 6 days, and the cell morphology was observed.

Table 1: brown adipocyte induced differentiation medium formula

The results are shown in FIG. 1, which show the cell morphology after induction culture of the steady GFP-expressing HEF cells, the monostable CEBP beta-expressing HEF cells, and the steady co-expressing PPARgamma and CEBP beta HEF cells, respectively, by the brown adipocyte-induced differentiation medium (since the monostable PPARgamma-expressing HEF cells were not visible after induction culture of the brown adipocyte-induced differentiation medium, no lipid droplet formation was seen), whereas the steady co-expressing CEBP beta-expressing HEF cells and the steady co-expressing PPARgamma and CEBP beta HEF cells were visible in the cells after induction culture of the brown adipocyte-induced differentiation medium (oil red staining result), whereas only a small amount of lipid droplet formation was visible after induction culture of the monostable CEBP beta-expressing HEF cells by the brown adipocyte-induced differentiation medium, compared to the results of the steady GFP-expressing HEF cells. The above results indicate that co-expression of both pparγ and CEBP β can work together, wherein CEBP β promotes conversion of HEF cells to brown adipocytes, pparγ promotes further differentiation and maturation of brown adipocytes, so that the constructed HEF cells stably co-expressing pparγ and CEBP β have a higher potential to differentiate into brown adipocytes.

Example 2: preparation of mature brown adipocyte-derived exosomes transdifferentiated HEF cells

This example aims at inducing the HEF cells stably co-expressing PPARgamma and CEBP beta, which were constructed as described in example 1 and have the potential to differentiate into brown adipocytes, to transdifferentiate into mature brown adipocytes, and preparing exosomes using the mature brown adipocytes, specifically comprising the following steps.

(2.1) HEF cells stably co-expressing PPARgamma and CEBP beta, which were obtained by constructing in example 1, were induced to differentiate in brown adipocyte-induced differentiation medium (shown in Table 1, the formulation specifically used in this example was 98% by volume of high sugar DMEM, 2% by volume of fetal bovine serum, 33. Mu.M of biotin, 0.5. Mu.M of insulin, 17. Mu.M of pantothenic acid, 0.10. Mu.M of DEX, 2. Mu.M of T3, 0.55mM of IBMX and 0.03mM of indomethacin) for 6 days.

(2.2) cells after induced differentiation in step (2.1) were washed 3 times with PBS, and 20mL of phenol red-free DMEM basal medium was added to each dish. After 24 hours of culture, the cell culture supernatant was collected, and the culture was continued for 24 hours by adding new 20mL of phenol red-free DMEM basal medium, and the cell culture supernatant was collected.

(2.3) the cell culture supernatant collected in (2.2) was placed in a centrifuge at 4℃and centrifuged at 1800rpm for 25min, and after centrifugation, the dead cell pellet was discarded and the supernatant was collected.

(2.4) the supernatant collected in (2.3) was placed in a centrifuge at 4℃and centrifuged at 9500rpm for 35min, and after centrifugation, the cell debris pellet was discarded and the supernatant was collected.

(2.5) the supernatant collected in (2.4) was placed in a centrifuge at 4℃and centrifuged at 100000 Xg for 50min, and the precipitate was collected after centrifugation.

(2.6) resuspension of the precipitate collected in step (2.5) with phenol red free DMEM to obtain brown adipocyte-derived exosomes. Quantitative protein analysis based on Label Free analysis of exosome samples, as shown in Table 2 below, revealed that the obtained exosomes contained a large amount of CD81 protein (marker protein of exosomes) but did not contain COX IV protein (marker protein of mitochondria), confirming that the exosomes were indeed obtained in this example.

Table 2: results of exosome marker protein abundance (intensity) analysis in samples

Example 3: use of brown adipocyte-derived exosomes in the treatment of mouse arthritis

This example was directed to treating a collagen-induced mouse arthritis model animal using brown adipocyte-derived exosomes prepared in example 2, and evaluating the efficacy, specifically comprising the following steps.

3.1A mouse model of collagen-induced arthritis (collagen-induced arthritis, CIA) was constructed using DBA/1J mice (Male, 8 week old, purchased from Beijing Vitre Lihua laboratory animal technologies Co., ltd., and fed to the institute of China academy of sciences animal management center, SPF grade, room temperature 24.+ -. 2 ℃ for 12 hours light/dark cycle) by the following specific method: bovine type II collagen (type II collagen, CII, chondrex inc 20021) was mixed with 50. Mu.l of adjuvant (Chondrex 7001) and fully emulsified, and the first immunization was performed by subcutaneous injection at the root of DBA/1J mice, and after 21 days 50. Mu. l C II was mixed with 50. Mu.l of incomplete adjuvant (Chondrex 7002) and fully emulsified for re-immunization by subcutaneous injection at the root of mice. Treatment was performed 21 days after the first immunization, specifically, mice were randomly divided into 2 groups (10 mice per group), wherein the first group was a control group, and vitamin E milk was applied once daily to the limbs of the mice (standard, home-made); another group was treated with vitamin E milk addition of brown adipocyte-derived exosomes prepared in example 2 (wherein the total protein concentration was about 1 mg/ml) and applied once daily on the four limbs of mice for 21 consecutive days. All animal experimental schemes above have been approved by the animal welfare ethics review committee of the national academy of sciences of China, and all experiments were performed strictly according to the animal ethics guidelines and approved animal experimental system designs.

3.2, index: the extent of swelling of the joints of the limbs (expressed as average thickness of paw swelling in mice) was observed and measured on day 21 (i.e., day of treatment, 0 w), day 28 (i.e., one week of treatment, 1 w), day 35 (i.e., two weeks of treatment, 2 w) and day 42 (i.e., three weeks of treatment, 3 w), respectively, and the serum of each group was collected on day 42 (i.e., three weeks of treatment, 3 w) and the levels of inflammatory factor TNFa in the serum of each group was detected using ELISA kit (CUSABIO, CSB-E04741 m). The knee joints of the mice of the control group and the exosome treatment group on day 42 were fixed for 48 hours, rinsed with running water for 2 hours, decalcified in 10% EDTA decalcified solution, replaced 2 times per week for 4 weeks, embedded in conventional paraffin, sectioned (6 μm) for later use, stained according to safranine fast green (Soy Bao G1371) instructions, sealed and observed with a microscope.

3.3 results

(1) The curves of the average paw swelling thickness over time for each group of mice measured from day 21 to day 42 (0 w-3 w) are shown in fig. 2, and it can be seen that paw redness occurred in each group of model mice on day 21, and from day 28 (one week of treatment, 1 w), the average paw swelling thickness was significantly lower for the exosome-treated group of mice than for the control group (P < 0.05); after two weeks of treatment, the average paw swelling thickness of the mice in the exosome treatment group was significantly lower than that of the control group (P < 0.01), wherein the average thickness of the control group at 2w was 3.23mm, and the average thickness of the exosome treatment group was 2.39mm; the average thickness of the control group at 3w was 3.28mm and the exosome treatment group was 2.64mm. The results show that the thickness of paw swelling of the mice can be obviously reduced when the mice with the model of collagen-induced arthritis are treated by external administration except for exosomes, and the mice have good effect of eliminating inflammation from appearance.

(2) The results of examination of the levels of the inflammatory factor TNFa in the serum of each group of mice are shown in FIG. 3, and it can be seen that the levels of the inflammatory factor TNFa in the serum of the mice of the exosome-treated group are significantly lower (P < 0.05) relative to the control group. The results show that when the external administration mode except for exosomes is used for treating the collagen-induced arthritis model mouse, the level of inflammatory factor TNFa in serum of the model mouse can be obviously reduced, and a good effect of eliminating inflammation is realized.

(3) Microscopic observations of paraffin embedded sections of knee joints of mice in the control group and the exosome treatment group are shown in fig. 4, and compared with knee joints of mice in the control group, cartilage surfaces at knee joints of mice in the exosome treatment group are smoother, inflammatory cell infection is obviously reduced, and the external application administration of exosomes derived from brown fat cells at knee joints is shown to have good improvement and treatment effects on arthritis symptoms of model mice.

The above results show that the brown adipocyte-derived exosomes (prepared in example 2) provided by the present invention can significantly improve their arthritic lesions indications by applying to the extremities of the CIA mouse model. In addition, compared with the injection mode reported in the prior art, which is invasive treatment, risks including local hemorrhage, hematoma, infection, local tissue embolism, local tissue necrosis and the like exist, the external medicine mode adopted by the invention is safer and more convenient.

Example 4: application of brown adipocyte-derived exosomes

This implementation was intended to treat patients with arthritis-related conditions using brown adipocyte-derived exosomes prepared in example 2, and to evaluate the efficacy, specifically comprising the following steps.

9 patients with arthritic pain (signed informed consent, specific information of the patients is shown in table 3 below) were selected for treatment with brown adipocyte-derived exosomes prepared in example 2 and subsequently subjected to medical evaluation. The specific treatment mode is as follows: the patient smears brown adipocyte-derived exosomes prepared in example 2 (wherein the total protein concentration was about 1 mg/ml) once a day in the morning and evening at the painful site of the patient for 14 consecutive days, and records the pain index, the evaluation method and criteria of which are as follows:

visual simulation method (VAS line drawing method)

A long line (typically 100 mm) is drawn between painless/severe pain, and no marks, numbers or words are made on the line so as not to affect the evaluation result. One end represents no pain and the other end represents severe pain, so that the patient can draw a cross line on the line where the pain degree can be reflected by the patient.

VAS pain scoring criteria (0 min-10 min)

0 point: no pain;

less than or equal to 3 minutes: slight pain, tolerance;

4-6: the patients are painful and influence sleep, and can endure;

more than or equal to 7 minutes: patients have gradually intense pain, which is hard to endure, affects appetite and affects sleep.

The evaluation results are shown in Table 3 below. It can be seen that the pain index of arthritis-related conditions including gouty arthritis, cervical disc herniation, lumbar disc degeneration and tendinitis can be significantly reduced after applying the exosomes provided by the present invention to the pain sites of arthritis-related conditions patients for 3 days, and the pain index of these arthritis-related conditions can be significantly reduced after applying the exosomes for 7 days (especially 14 days later), and the exosomes provided by the present invention have better therapeutic effects (the pain index is generally reduced from 7-9 to 0-2) on gouty arthritis and cervical disc herniation among these arthritis-related conditions, and the pain of patients can be substantially completely relieved after applying the exosomes for 14 days. The results prove that the HEF cell transdifferentiated brown adipocyte-derived exosomes prepared by the invention have good effect of relieving pain of arthritis-related diseases.

Table 3: evaluation of therapeutic Effect of brown adipocyte-derived exosomes on arthritis-related disorders

Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of brown adipocyte-derived exosomes in the manufacture of a medicament for the treatment of an arthritis-related disorder; wherein:

the medicine is an external medicine;

2. The use according to claim 1, wherein said operation of constructing human skin fibroblasts co-expressing pparγ and CEBP β in step S1 is:

3. The use according to claim 1 or 2, wherein the coding sequences of pparγ and cebpβ in step S1 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

4. The use according to claim 1 or 2, wherein the formulation of the brown adipocyte-induced differentiation medium in step S2 comprises: 97-99% by volume of high sugar DMEM, 1-3% by volume of fetal bovine serum, 32-34. Mu.M biotin, 0.4-0.6. Mu.M insulin, 16-18. Mu.M pantothenic acid, 0.09-0.11. Mu.M DEX, 1-3. Mu.M T3, 0.50-0.60mM IBMX and 0.02-0.04mM indomethacin.

5. The use according to claim 1 or 2, wherein the time for inducing differentiation culture in step S2 is 6 days or more.

6. The use according to claim 1 or 2, wherein the conditions for inducing differentiation culture in step S2 are 5% co ₂ ，37±1℃。

7. The use according to claim 1 or 2, wherein the step S3 of secreting exosomes from the brown adipocytes obtained in step S2 is performed by culturing the brown adipocytes using a serum-free basal medium.

8. A medicament for treating arthritis-related conditions comprising as an active ingredient the brown adipocyte-derived exosomes mentioned in any one of claims 1 to 7;

the medicine is an external medicine.

9. The medicament of claim 8, wherein the dosage form of the medicament comprises powder, solution, tincture, spirit, lotion, ointment, cream, paste, oil, gel, film coating agent, liniment, drops, aerosol, wet dressing, suppository, patch and spray.

10. The medicament according to claim 8 or 9, wherein the arthritis-related conditions comprise gouty arthritis, cervical disc herniation, lumbar disc lesions and tendinitis, preferably gouty arthritis and cervical disc herniation.