Composition for treating degenerative arthritis and application thereof

Abstract

The invention belongs to the technical field of stem cell treatment, and discloses a composition for treating degenerative arthritis and application of the composition, and the composition comprises the following components in percentage by mass: 20-60% of adipose-derived stem cells, 10-30% of bone marrow mesenchymal stem cells and 10-50% of traditional Chinese medicines. According to the application, the adipose-derived stem cells with stronger multiplication capacity are obtained by improving a culture medium of the adipose-derived stem cells, miRNA-140 of the adipose-derived stem cells is highly expressed, inflammatory factors can be effectively inhibited after the adipose-derived stem cells are compounded with bone marrow mesenchymal stem cells and turmeric powder in proportion, and the recovery of cartilage tissues is accelerated.

Description

Technical Field

[0001] The present application relates to the technical field of stem cell therapy, and in particular to a composition for treating degenerative arthritis and its application. Background Art

[0002] Osteoarthritis, also known as osteoarthritis, hyperplastic arthritis, senile arthritis, degenerative arthritis, hypertrophic arthritis, etc., is a chronic joint disease, the main changes of which are degenerative disease of articular cartilage and secondary bone hyperplasia. According to the pathogenic factors, it is divided into primary osteoarthritis and secondary osteoarthritis. Any normal joint that gradually degenerates without obvious reasons is called primary osteoarthritis; if the cartilage is destroyed or the joint structure is changed due to some known reasons, and the degeneration caused by factors such as friction of the joint surface or pressure imbalance in the future is called secondary osteoarthritis.

[0003] Mesenchymal stem cells (MSCs) are a type of multipotent adult stem cells with stem cell characteristics and are considered to be a promising cell therapy for the treatment of knee osteoarthritis.

[0004] For example, the prior art CN 117959350 B discloses an osteoarthritis treatment composition based on a composite stem cell SVF, which is composed of the following components: 40-50% by mass of SVF, 16-20% by mass of bone marrow mesenchymal stem cells and a Chinese medicine extract making up the balance, wherein the Chinese medicine comprises angelica sinensis, 14-18% by mass of safflower, 24-30% by mass of Panax notoginseng and a balance of Tripterygium wilfordii.

[0005] Prior art CN 117815335 B discloses an osteoarthritis treatment composition based on adipose-derived stem cell SVF and a preparation method thereof, wherein the osteoarthritis treatment composition based on adipose-derived stem cell SVF is composed of the following components: 60-70% by weight of adipose-derived stem cell SVF and a Chinese medicine extract that makes up the balance. The adipose-derived stem cell SVF is combined with the Chinese medicine extract unique to the present invention to effectively eliminate inflammatory factors while promoting the recovery of bone and joint cartilage, and the Chinese medicine contains angelica, Lysimachia chinensis, Dendrobium officinale and Coptis chinensis.

[0006] Prior art CN 118221761 B discloses a stem cell composition for treating osteoarthritis, characterized in that the composition comprises 1×10 6 -1×10 9 cells / mL human umbilical cord mesenchymal stem cells, 10-20ng / ml directed differentiation-inducing polypeptide and 100-200μg / mL chondroitin sulfate.

[0008] Acquiring stem cells with strong survival, migration and differentiation abilities is particularly important for therapeutic effects. Therefore, the present application aims to develop a more effective stem cell culture method and a composition for treating degenerative arthritis comprising the stem cells. Summary of the invention

[0009] In order to solve the above problems, the purpose of the present invention is to provide a composition for treating degenerative arthritis, characterized in that the composition for treating degenerative arthritis consists of the following components: adipose stem cells, bone marrow mesenchymal stem cells and traditional Chinese medicine; the adipose stem cells are cultured in a culture medium containing 10% FBS (fetal bovine serum), low-glucose DMEM, 10ng / ml SCF, 50ng / ml glutathione and 60ng / ml VEGF, and can highly express miRNA-140.

[0010] In certain embodiments, the composition comprises 20-60% by weight of adipose stem cells, 10-30% by weight of bone marrow mesenchymal stem cells, and 10-50% by weight of traditional Chinese medicine.

[0011] In certain embodiments, the traditional Chinese medicine is turmeric powder.

[0012] In certain embodiments, the mass fraction of the adipose stem cells is 40-50%.

[0013] In certain embodiments, the mass fraction of the bone marrow mesenchymal stem cells is 15-25%.

[0014] In certain embodiments, the mass fraction of the turmeric powder is 10-30%.

[0015] The present invention also provides an application of the above composition for treating degenerative arthritis in preparing a medicine for treating degenerative arthritis.

[0016] This application can bring the following beneficial effects:

[0017] The present application improves the culture medium of adipose stem cells to obtain adipose stem cells with stronger proliferation ability, and their miRNA-140 is highly expressed. After being compounded with bone marrow mesenchymal stem cells and turmeric powder in proportion, they can effectively inhibit inflammatory factors and accelerate the recovery of cartilage tissue. DETAILED DESCRIPTION

[0018] The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0019] The reagents used in this application were purchased from commercial companies.

[0020] Example 1: Preparation of the culture medium used in the present invention:

[0021] Medium 1: low glucose DMEM with 10% FBS (fetal bovine serum); 10 ng / ml SCF, 50 ng / ml glutathione and 60 ng / ml VEGF.

[0022] Medium 2: low-glucose DMEM containing 10% FBS (fetal bovine serum); 10 ng / ml SCF, 50 ng / ml glutathione.

[0023] Medium 3: low-glucose DMEM with 10% FBS (fetal bovine serum); 10 ng / ml SCF and 60 ng / ml VEGF.

[0024] Medium 4: low-glucose DMEM with 10% FBS (fetal bovine serum); 50 ng / ml glutathione and 60 ng / ml VEGF.

[0025] Medium 5: low-glucose DMEM containing 10% FBS (fetal bovine serum); 10 ng / ml SCF.

[0026] Medium 6: low-glucose DMEM containing 10% FBS (fetal bovine serum); 50 ng / ml glutathione.

[0027] Culture medium 7: low-glucose DMEM containing 10% FBS (fetal bovine serum); 60 ng / ml VEGF.

[0028] Medium 8: low-glucose DMEM containing 10% FBS (fetal bovine serum).

[0029] Example 2 A method for preparing adipose-derived stem cells,

[0030] The following steps are involved:

[0031] (1) Under sterile conditions, 5 g of human adipose tissue was obtained, washed with PBS buffer, and minced to obtain adipose tissue homogenate, which was then dispensed into 50 ml centrifuge tubes.

[0032] (2) Add equal volumes of type I collagenase and trypsin to the centrifuge tube in step (1), incubate in a 37°C water bath, and digest for 60 min. After inactivation of the enzymes, obtain adipose tissue enzymatic hydrolysate.

[0033] (3) The resulting enzymatic hydrolysate was centrifuged at 2500 rpm for 15 minutes, the cell precipitate was collected, and the adipose stem cell cluster at the bottom was retained.

[0034] (4) Add 10 ml of PBS buffer to the centrifuge tube containing the adipose stem cell cluster, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a filter with a diameter of 100 μm, collect single cells, and then centrifuge at 2500 rpm for 10 minutes, discard the upper liquid, and retain the adipose stem cell cluster at the bottom; repeat step (4) twice.

[0035] (5) After sufficient washing, add an appropriate amount of physiological saline, repeatedly blow the cells to make them into a single cell suspension of adipose stem cells, add them to a culture bottle containing culture medium 1, and place them in a 5% CO2 concentration 37°C incubator for culture; after culturing for 2 days, first replace culture medium 1 to remove non-adherent cells, and then replace the culture medium every 3 days until the cell density reaches about 80% confluence, and pass them at a ratio of 1:3, and take the third generation of adipose stem cells for the experiment. The surface antigen detection of the third generation of cultured adipose stem cells showed that adipose stem cell markers such as CD29, CD49, CD73, CD90 and CD105 were expressed, and CD34, CD45 and HLA-DR were not expressed, proving that the cells are adipose stem cells.

[0036] Comparative Example 1

[0037] Except that medium 2 was used to replace medium 1, other conditions were the same as those in Example 2.

[0038] Comparative Example 2

[0039] Except that medium 3 was used to replace medium 1, other conditions were the same as those in Example 2.

[0040] Comparative Example 3

[0041] Except that medium 4 was used to replace medium 1, other conditions were the same as those in Example 2.

[0042] Comparative Example 4

[0043] Except that medium 5 was used to replace medium 1, other conditions were the same as those in Example 2.

[0044] Comparative Example 5

[0045] Except that medium 6 was used to replace medium 1, other conditions were the same as those in Example 2.

[0046] Comparative Example 6

[0047] Except that medium 7 was used to replace medium 1, other conditions were the same as those in Example 2.

[0048] Comparative Example 7

[0049] Except that medium 8 was used to replace medium 1, other conditions were the same as those in Example 2.

[0050] Example 3 Detection of Adipose Stem Cells

[0051] The adipose stem cells were cultured and subcultured to the 10th generation (P10) using the culture methods of Example 2 and Comparative Examples 1-7, and the cells were counted using the trypan blue classic staining method, observed and counted under a microscope, and the cell viability was calculated based on the staining conditions. Based on the cell number of Comparative Example 7, the relative cell number of each group was calculated, and the results are shown in Table 1.

[0052] Table 1 Relative cell number of adipose stem cells cultured and expanded in vitro

[0053] Group Relative number of P10 Example 2 30 Comparative Example 1 12 Comparative Example 2 11 Comparative Example 3 12 Comparative Example 4 10 Comparative Example 5 9 Comparative Example 6 9 Comparative Example 7 1

[0054] The cell counting results are shown in Table 1. The culture medium 1 of Example 2 has the best effect, and compared with the control (Comparative Example 7), the effect has a very significant difference (p<0.01), indicating that the culture medium 1 can significantly promote the proliferation of adipose stem cells.

[0055] At the same time, the differentiation potential of P10 adipose stem cells was detected. The results of Oil Red O staining and silver nitrate staining showed that P10 adipose stem cells still had obvious adipogenic and osteogenic abilities.

[0056] The prior art has found that miRNA-140 inhibition can destroy articular chondrocytes during the development of osteoarthritis, and high expression of miRNA-140 can inhibit OA chondrocyte apoptosis and promote its proliferation (see CN 111494707A, and Cai Feng et al., Effect of MiRNA-140 Inhibition on the Treatment of Osteoarthritis, Jiangxi Medicine, December 2021, Vol. 56, No. 12). Therefore, total RNA extraction, reverse transcription and real-time quantitative PCR (qPCR) were performed on P10 adipose stem cells to detect the expression of miRNA-140 in adipose stem cells of Example 2 and Comparative Examples 1 to Comparative Example 7, and the relative expression of each group was calculated based on the expression of Comparative Example 7, and the results are shown in Table 2. The relative expression of miRNA-140 in Example 2 was significantly improved, indicating that it has a stronger ability to treat osteoarthritis (p<0.01).

[0057] Table 2 Relative expression of miRNA-140 in each group

[0058]

[0059]

[0060] Example 4 Preparation of bone marrow mesenchymal stem cells:

[0061] Autologous bone marrow was collected from the iliac bone using a bone marrow puncture needle. The amount of bone marrow collected each time was 15 ml. The samples were centrifuged at 1500 r / min for 10 min, the upper fat tissue was removed, and the precipitate was suspended with an equal mass of PBS. The same mass of human lymphocyte separation solution was injected and centrifuged at 1500 r / min for 20 min. After centrifugation, the intermediate mononuclear cells were taken and washed twice with PBS. The mononuclear cells were aspirated and resuspended in 30 times the mass of human mesenchymal stem cell conditioned medium. The cell density was adjusted to 2×10 5 The cells were inoculated into culture flasks at 37°C and 5% carbon dioxide incubator for 48 h, and then washed with saline. The cell density was adjusted to 1×10 5 / mL, and obtain the bone marrow mesenchymal stem cells;

[0062] Example 5

[0063] 40% of the adipose-derived stem cells (prepared in Example 2, 1×10 5 / mL), bone marrow mesenchymal stem cells with a mass fraction of 20% (prepared in Example 4, 1×10 5 / mL) and 40% by mass of turmeric powder are mixed to prepare a composition for treating degenerative arthritis.

[0064] The difference between Comparative Example 8 and Example 5 is that the composition does not contain turmeric powder, and the other conditions are the same;

[0065] The difference between Comparative Example 9 and Example 5 is that the composition does not contain adipose stem cells, and the other conditions are the same;

[0066] The difference between Comparative Example 10 and Example 5 is that the composition does not contain bone marrow mesenchymal stem cells, and the other conditions are the same;

[0067] The difference between Comparative Example 11 and Example 5 is that the composition contains only Chinese medicine, and the other conditions are the same;

[0068] The difference between Comparative Example 12 and Example 5 is that the composition contains only adipose stem cells, and the other conditions are the same;

[0069] The only difference between Comparative Example 13 and Example 5 is that the composition contains only bone marrow mesenchymal stem cells, and the other conditions are the same.

[0070] Take the composition for treating degenerative arthritis obtained in each example and slowly inject 4 mL of the composition into the joints of each person suffering from osteoarthritis of the same degree using a syringe. The same degree is based on the proportion of cartilage loss of 20% in the affected part of the subject observed by magnetic resonance imaging MRI. There are 24 subjects, divided into 8 groups, 3 people in each group. Inject the composition for treating degenerative arthritis obtained in each example respectively, and inject it again in the second and third months respectively. At the 35th week after treatment, the blank group is not treated and the following tests are performed;

[0071] Immune response testing:

[0072] The amount of IL-1β inflammatory factor characterizes the level of immune response, and the two are positively correlated. The synovial fluid was collected and the expression of IL-1β inflammatory factor was detected by ELISA. The content detection is shown in Table 3. The unit of the test result is pg / ml, and the integer is retained.

[0073] Cartilage recovery test:

[0074] The joints of the patients were observed by magnetic resonance imaging (MRI) to detect the recovery of the joints, i.e., the area of ​​cartilage loss. The area of ​​articular cartilage loss after 35 weeks of treatment is shown in Table 1, in %, with integers retained.

[0075] The above test results are taken as the average of 3 people in each sample and blank group and the integer is retained.

[0076] Table 3: Results of immune response test and cartilage restoration test for each example

[0077] Example IL-1β content (pg / ml) Cartilage loss area (%) Example 5 329 2 Comparative Example 8 711 5 Comparative Example 9 950 7 Comparative Example 10 857 6 Comparative Example 11 1597 12 Comparative Example 12 910 7 Comparative Example 13 1231 10 Blank Group 9874 18

[0078] It can be seen from the data in Table 3 that Example 5 has a lower IL-1β content and a lower cartilage loss area (p<0.01) compared with the blank group or other control groups. Example 5 of the present application has the best ability to treat osteoarthritis.

[0079] The above description is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of the claims of the present application.

Claims

1. A composition for treating degenerative arthritis, characterized in that: The composition for treating degenerative arthritis is composed of the following components: adipose stem cells, bone marrow mesenchymal stem cells and traditional Chinese medicine; The adipose stem cells are prepared by culturing with a culture medium containing 10% FBS (fetal bovine serum) low-glucose DMEM, 10 ng / ml SCF, 50 ng / ml glutathione and 60 ng / ml VEGF, and can highly express miRNA-140.

2. The composition for treating degenerative arthritis according to claim 1, characterized in that: The composition comprises 20-60% by weight of adipose stem cells, 10-30% by weight of bone marrow mesenchymal stem cells and 10-50% by weight of traditional Chinese medicine.

3. The composition for treating degenerative arthritis according to claim 1, characterized in that: The traditional Chinese medicine is turmeric powder.

4. The composition for treating degenerative arthritis according to claim 1, characterized in that: The mass fraction of the adipose stem cells is 40-50%.

5. The composition for treating degenerative arthritis according to claim 1, characterized in that: The mass fraction of the bone marrow mesenchymal stem cells is 15-25%.

6. The composition for treating degenerative arthritis according to claim 1, characterized in that: The mass fraction of the turmeric powder is 10-30%.

7. Use of the composition for treating degenerative arthritis according to any one of claims 1 to 6 in the preparation of a medicament for treating degenerative arthritis.

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