A composition for treating degenerative arthritis and its applications

Abstract

This application belongs to the technical field of stem cell therapy, and discloses a composition for treating degenerative arthritis and its application. The composition contains adipose stem cells in a mass fraction of 20-60%, bone marrow mesenchymal stem cells in a mass fraction of 10-30%, and traditional Chinese medicine in a mass fraction of 10-50%. By improving the culture medium of adipose stem cells, this application obtains adipose stem cells with stronger proliferation ability, and its miRNA-140 is highly expressed. After being compounded with bone marrow mesenchymal stem cells and turmeric powder in proportion, it can effectively inhibit inflammatory factors and accelerate the recovery of cartilage tissue.

Description

Technical Field

[0001] This application relates to the technical field of stem cell therapy, and particularly to a composition for treating degenerative arthritis and its applications. Background Art

[0002] Osteoarthrosis, also known as osteoarthritis, proliferative arthritis, senile arthritis, degenerative arthritis, hypertrophic arthritis, etc., is a chronic joint disease. Its main changes are the degeneration of articular cartilage and secondary osteophytosis. According to the causative factors, it is divided into primary osteoarthrosis and secondary osteoarthrosis. When normal joints gradually undergo degeneration without obvious reasons, it is called primary osteoarthrosis; if cartilage damage or joint structure changes are caused by a certain known reason, and then degenerative changes occur due to factors such as joint surface friction or pressure imbalance in the future, it is called secondary osteoarthrosis.

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

[0004] For example, the prior art CN 117959350 B discloses an osteoarthritis treatment composition based on composite stem cell SVF. The osteoarthritis treatment composition based on composite stem cell SVF consists of the following components: 40 - 50% by mass of SVF, 16 - 20% by mass of bone marrow mesenchymal stem cells, and the balance of traditional Chinese medicine extract. The traditional Chinese medicine includes Angelica sinensis, 14 - 18% by mass of Carthamus tinctorius, 24 - 30% by mass of Panax notoginseng, and the balance of Tripterygium wilfordii.

[0005] The prior art CN 117815335 B discloses an osteoarthritis treatment composition based on adipose stem cell SVF and its preparation method. The osteoarthritis treatment composition based on adipose stem cell SVF consists of the following components: 60 - 70% by weight of adipose stem cell SVF and the balance of traditional Chinese medicine extract. By combining adipose stem cell SVF with the unique traditional Chinese medicine extract of the present invention, while promoting the recovery of articular cartilage of bones and joints, inflammatory factors can be effectively eliminated. The traditional Chinese medicine includes Angelica sinensis, Lysimachia christinae, Dendrobium nobile, and Coptis chinensis.

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

[0007] It is particularly important to obtain stem cells with strong survival, migration, and differentiation abilities for the treatment effect. Therefore, the present application aims to develop a more effective stem cell culture method and a composition for treating degenerative arthritis containing such stem cells. Summary of the Invention

[0008] To solve the above problems, the object of the present invention is to provide a composition for treating degenerative arthritis, which is 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 cultured and prepared using a culture medium of low-glucose DMEM with 10% FBS (fetal bovine serum), 10 ng / ml SCF, 50 ng / ml glutathione, and 60 ng / ml VEGF, and can highly express miRNA-140.

[0009] In certain embodiments, the composition has a mass fraction of 20-60% adipose stem cells, a mass fraction of 10-30% bone marrow mesenchymal stem cells, and 10-50% traditional Chinese medicine.

[0010] In certain embodiments, the traditional Chinese medicine is turmeric powder.

[0011] In certain embodiments, the mass fraction of the adipose stem cells is 40-50%.

[0012] In certain embodiments, the mass fraction of the bone marrow mesenchymal stem cells is 15-25%.

[0013] In certain embodiments, the mass fraction of the turmeric powder is 10-30%.

[0014] The present invention also provides an application of the above composition for treating degenerative arthritis in the preparation of a drug for treating degenerative arthritis.

[0015] The present application can bring the following beneficial effects:

[0016] By improving the culture medium of adipose stem cells, the present application obtains adipose stem cells with stronger proliferation ability, and its miRNA-140 is highly expressed. After being compounded with bone marrow mesenchymal stem cells and turmeric powder in proportion, it can effectively inhibit inflammatory factors and accelerate the recovery of cartilage tissue. Detailed Embodiments

[0017] The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to 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.

[0018] The reagents of the present application are all purchased from commercial companies.

[0019] Example 1. Preparation of the culture medium used in the present invention:

[0020] Culture medium 1: Low-glucose DMEM with 10% FBS (fetal bovine serum); 10 ng / ml SCF, 50 ng / ml glutathione, and 60 ng / ml VEGF.

[0021] Culture medium 2: Low-glucose DMEM with 10% FBS (fetal bovine serum); 10 ng / ml SCF, 50 ng / ml glutathione.

[0022] Culture medium 3: Low-glucose DMEM with 10% FBS (fetal bovine serum); 10 ng / ml SCF and 60 ng / ml VEGF.

[0023] Culture medium 4: Low-glucose DMEM with 10% FBS (fetal bovine serum); 50 ng / ml glutathione and 60 ng / ml VEGF.

[0024] Culture medium 5: Low-glucose DMEM with 10% FBS (fetal bovine serum); 10 ng / ml SCF.

[0025] Culture medium 6: Low-glucose DMEM with 10% FBS (fetal bovine serum); 50 ng / ml glutathione.

[0026] Culture medium 7: Low-glucose DMEM with 10% FBS (fetal bovine serum); 60 ng / ml VEGF.

[0027] Culture medium 8: Low-glucose DMEM with 10% FBS (fetal bovine serum).

[0028] Example 2. A method for preparing adipose stem cells,

[0029] comprising the following steps:

[0030] (1) Under sterile conditions, 5 g of human adipose tissue was obtained, washed with PBS buffer, minced, and then homogenized to obtain adipose tissue homogenate, which was aliquoted into 50 ml centrifuge tubes.

[0031] (2) Equal volumes of type I collagenase and trypsin were added to the centrifuge tubes in step (1), and the mixture was incubated in a water bath at 37°C for 60 min, followed by inactivation of the enzymes to obtain adipose tissue enzymatic hydrolysate.

[0032] (3) The obtained enzymatic hydrolysate was centrifuged at a speed of 2500 revolutions per minute for 15 minutes, and the cell pellet was collected to retain the adipose stem cell mass at the bottom layer.

[0033] (4) Add 10 ml of PBS buffer to the centrifuge tube containing the adipose-derived stem cell mass, pipette repeatedly to resuspend the cells to form a uniform single-cell suspension, wash thoroughly, pass the cells through a filter with a diameter of 100 microns, collect single cells, and then centrifuge at a speed of 2500 revolutions per minute for 10 minutes. Discard the supernatant and retain the adipose-derived stem cell mass at the bottom; repeat step (4) twice.

[0034] (5) After thorough washing, add an appropriate amount of normal saline, pipette the cells repeatedly to form a single-cell suspension of adipose-derived stem cells, add it to the culture flask containing Medium 1, and place it in an incubator at 37°C with a 5% CO2 concentration for culture; after 2 days of culture, first replace Medium 1 to remove non-adherent cells, and then replace the medium every 3 days until the cell density reaches about 80% confluence, and passage at a ratio of 1:3. Take the third-generation adipose-derived stem cells for experiments. Perform surface antigen detection on the third-generation adipose-derived stem cells in culture. Results: Markers of adipose-derived stem cells such as CD29, CD49, CD73, CD90, and CD105 are expressed, while CD34, CD45, and HLA-DR are not expressed, proving that the cells are adipose-derived stem cells.

[0035] Comparative Example 1

[0036] Except for using Medium 2 to replace Medium 1, the other conditions are the same as those in Example 2.

[0037] Comparative Example 2

[0038] Except for using Medium 3 to replace Medium 1, the other conditions are the same as those in Example 2.

[0039] Comparative Example 3

[0040] Except for using Medium 4 to replace Medium 1, the other conditions are the same as those in Example 2.

[0041] Comparative Example 4

[0042] Except for using Medium 5 to replace Medium 1, the other conditions are the same as those in Example 2.

[0043] Comparative Example 5

[0044] Except for using Medium 6 to replace Medium 1, the other conditions are the same as those in Example 2.

[0045] Comparative Example 6

[0046] Except for using Medium 7 to replace Medium 1, the other conditions are the same as those in Example 2.

[0047] Comparative Example 7

[0048] Except for using Medium 8 to replace Medium 1, the other conditions are the same as those in Example 2.

[0049] Example 3 Detection of adipose-derived stem cells

[0050] Take the adipose-derived stem cells cultured by the culture methods of Example 2 and Comparative Examples 1-7 and passaged to the 10th generation (P10). Use the classic trypan blue staining method to count the cells, place them under a microscope to observe and count, and calculate the cell viability according to the staining situation. And based on the cell number of Comparative Example 7, calculate the relative cell numbers of each group. The results are shown in Table 1.

[0051] Table 1 Relative cell numbers of adipose-derived stem cells in vitro culture and expansion

[0052] Group Relative quantity 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

[0053] The cell counting results are shown in Table 1. The culture medium 1 of Example 2 has the best effect. Compared with the control (Comparative Example 7), this effect has a highly significant difference (p < 0.01). It shows that the above-mentioned culture medium 1 can significantly promote the proliferation of adipose-derived stem cells.

[0054] At the same time, detect the differentiation potential of adipose-derived stem cells at the P10 generation. The results of oil red O staining and silver nitrate staining show that the adipose-derived stem cells at P10 still have obvious adipogenic ability and osteogenic ability.

[0055] It has been found in the prior art that miRNA-140 inhibition can damage articular chondrocytes during the occurrence and development of osteoarthritis, and high expression of miRNA-140 can inhibit the apoptosis of OA chondrocytes and promote their proliferation (see CN 111494707A, and Cai Feng et al., The therapeutic effect of miRNA-140 inhibition on osteoarthritis, Jiangxi Medical Journal, Vol. 56, No. 12, December 2021). Therefore, total RNA extraction, reverse transcription and real-time quantitative PCR (qPCR) were performed on P10 adipose-derived stem cells to detect the expression levels of miRNA-140 in the adipose-derived stem cells of Example 2 and Comparative Examples 1-7. And based on the expression level of Comparative Example 7, calculate the relative expression levels of each group. The results are shown in Table 2. The relative expression level of miRNA-140 in Example 2 was significantly increased, indicating that it has stronger osteoarthritis treatment ability (p < 0.01).

[0056] Table 2 Relative expression levels of miRNA-140 in each group

[0057]

[0058]

[0059] Example 4 Preparation of bone marrow mesenchymal stem cells:

[0060] Autologous bone marrow was collected from the ilium using a bone marrow puncture needle. Each collection volume was 15 ml, and it was centrifuged at 1500 r / min for 10 min. The upper adipose tissue was aspirated, and after suspending the precipitate with an equal mass of PBS, an equal mass of human lymphocyte separation solution was injected, and then centrifuged at 1500 r / min for 20 min. After centrifugation, the middle mononuclear cells were taken and washed twice with PBS, and 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 cells / ml and inoculated into a culture flask. After culturing in an incubator at 37°C and 5% carbon dioxide for 48 h, physiological saline was added to wash the cells, and the cell density was adjusted to 1×10 5 cells / mL to obtain the bone marrow mesenchymal stem cells;

[0061] Example 5

[0062] A composition for treating degenerative arthritis was prepared by mixing 40 parts by mass of adipose stem cells (prepared in Example 2, 1×10 5 cells / mL), 20 parts by mass of bone marrow mesenchymal stem cells (prepared in Example 4, 1×10 5 cells / mL) and 40 parts by mass of turmeric powder.

[0063] The difference between Comparative Example 8 and Example 5 was only that the composition did not contain turmeric powder, and the other conditions were the same;

[0064] The difference between Comparative Example 9 and Example 5 was only that the composition did not contain adipose stem cells, and the other conditions were the same;

[0065] The difference between Comparative Example 10 and Example 5 was only that the composition did not contain bone marrow mesenchymal stem cells, and the other conditions were the same;

[0066] The difference between Comparative Example 11 and Example 5 was only that the composition only contained traditional Chinese medicine, and the other conditions were the same;

[0067] The difference between Comparative Example 12 and Example 5 was only that the composition only contained adipose stem cells, and the other conditions were the same;

[0068] The difference between Comparative Example 13 and Example 5 was only that the composition only contained bone marrow mesenchymal stem cells, and the other conditions were the same.

[0069] For the subjects aged 65 with osteoarthritis of the same degree, the compositions for treating degenerative arthritis obtained from each example were slowly injected into the joints affected by osteoarthritis of each person with a syringe at a rate of 4 mL. The same degree was based on the cartilage loss ratio of 20% observed by magnetic resonance imaging (MRI) at the affected area of the subjects. There were 24 subjects, divided into 8 groups with 3 people in each group. The compositions for treating degenerative arthritis obtained from each example were injected respectively, and then injected again in the second and third months. At the 35th week after treatment, the blank group was not treated, and the following tests were carried out;

[0070] Immune response test:

[0071] The level of the IL-1β inflammatory factor was used to characterize the level of the immune response, and the two were positively correlated; synovial fluid was taken, and the expression of the IL-1β inflammatory factor was detected by ELISA method. The content detection results are shown in Table 3, and the unit of the detection results is pg / ml, rounded to the nearest integer.

[0072] Cartilage recovery test:

[0073] The joints of the patients were observed by magnetic resonance imaging (MRI) to detect the joint recovery status, that is, the cartilage loss area. After 35 weeks of treatment, the cartilage loss area of the joints is shown in Table 1, and the unit is %, rounded to the nearest integer.

[0074] The above test results were the average values of 3 people in each example and the blank group, rounded to the nearest integer.

[0075] Table 3: Results of immune response test and cartilage recovery test for each example

[0076] Example IL-1β content (pg / ml) Cartilage defect 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

[0077] It can be seen from the data in Table 3 that Example 5 has lower IL-1β content and lower cartilage loss area compared with the blank group or other comparative examples (p < 0.01). Example 5 of the present application has the best ability to treat osteoarthritis specifically.

[0078] The above are only the embodiments of the present application and are not used to limit the present application; for those skilled in the art, various changes and modifications can be made to the present application; any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within 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 by mass fraction: 20-60% adipose stem cells, 10-30% bone marrow mesenchymal stem cells, and 10-50% turmeric powder; The adipose stem cells are prepared by culturing in a 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; The preparation method of the bone marrow mesenchymal stem cells is as follows: Autologous bone marrow was collected from the ilium through a bone marrow puncture needle. The amount collected each time was 15 ml. It was centrifuged at 1500 r / min for 10 min, and the upper adipose tissue was aspirated. After suspending the precipitate with an equal mass of PBS, an equal mass of human lymphocyte separation solution was injected, and it was centrifuged at 1500 r / min for 20 min. After centrifugation, the middle mononuclear cells were taken and washed 2 times with PBS, and 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 cells / ml and inoculated into a culture flask. After culturing in an incubator at 37°C and 5% carbon dioxide for 48 h, normal saline was added to wash the cells, and the cell density was adjusted to 1×10 5 cells / mL to obtain the bone marrow mesenchymal stem cells.

2. The composition for treating degenerative arthritis according to claim 1, wherein The mass fraction of the adipose stem cells is 40-50%.

3. The composition for treating degenerative arthritis according to claim 1, wherein The mass fraction of the bone marrow mesenchymal stem cells is 15-25%.

4. The composition for treating degenerative arthritis according to claim 1, wherein The mass fraction of the turmeric powder is 10-30%.

5. Use of a composition for treating degenerative arthritis according to any one of claims 1-4 in the preparation of a drug for treating degenerative arthritis.

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