Mesenchymal stem cell-containing composition for treating degenerative arthritis and preparation method thereof

Abstract

The invention relates to a composition containing mesenchymal stem cells for treating degenerative arthritis and a preparation method thereof. According to the application, the mesenchymal stem cells and the OSCAR antibody are combined for treating the degenerative arthritis for the first time, verification analysis shows that the mesenchymal stem cells and the OSCAR antibody have a synergistic effect, and the combination of the mesenchymal stem cells and the OSCAR antibody can be used for inhibiting secretion of inflammatory factors such as IL-1beta, TNF-alpha and IL-6 in a subject, relieving inflammatory injury of arthritis and preventing and treating the degenerative arthritis. The repair of arthritis can be improved and promoted. And after administration treatment, the symptoms of limb swelling, stiffness and the like of a subject are obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of degenerative arthritis treatment, and in particular to a composition comprising mesenchymal stem cells for treating degenerative arthritis and a preparation method thereof. Background Art

[0002] Degenerative arthritis is caused by the wear and tear of joints over the years. This disease often occurs in weight-bearing joints, such as the cervical spine, lumbar spine, knee joints, hip joints, heels, etc., so long-term or excessive weight-bearing should be avoided. Cold and dampness are important factors that cause the disease. This disease can occur in joints throughout the body, but it is more common in the knee joints, hip joints, spine and finger joints, which are heavy-bearing. Knee and hip joints are particularly prone to lesions.

[0003] Mesenchymal stem cells are a heterogeneous cell group derived from the matrix, which can be obtained from most tissues of the human body. A large number of experimental studies have shown that mesenchymal stem cells have the potential to differentiate into epidermal cells and can promote the healing of injured skin. Bone marrow-derived mesenchymal stem cells are not suitable for mass production because of the risk of viral contamination and the obvious downward trend in the number of cells and the ability to expand and differentiate as the donor ages. Mesenchymal stem cells derived from human umbilical cord, placenta or amniotic membrane can avoid many restrictions such as the lack of embryonic stem cell sources, allogeneic rejection, and moral ethics, and have become a good substitute for bone marrow-derived mesenchymal stem cells. Mesenchymal stem cells have the ability to self-renew, repair tissues, and regulate immunity. They can differentiate into the mesoderm lineage, such as into adipocytes, osteocytes, and chondrocytes. In addition, they can also differentiate into cells of other germ layer lineages, such as epidermal cells and vascular endothelial cells. Moreover, mesenchymal stem cells are easy to expand in vitro, and their differentiation and proliferation abilities remain stable after expansion, making them suitable for large-scale production.

[0004] Therefore, there is an urgent need for a safe therapeutic agent with excellent therapeutic effect to achieve the goal of treating degenerative arthritis. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a composition comprising mesenchymal stem cells for treating degenerative arthritis and a preparation method thereof. The present invention combines mesenchymal stem cells with OSCAR antibodies for the first time for the treatment of degenerative arthritis. After verification and analysis, it is found that there is a synergistic effect between mesenchymal stem cells and OSCAR antibodies. The combination of the two can be used to inhibit the secretion of inflammatory factors such as IL-1β, TNF-ɑ and IL-6 in the subject, relieve the inflammatory damage of arthritis, and improve and promote the repair of arthritis. After administration, the symptoms of swelling and stiffness of the limbs of the subject were significantly improved.

[0006] One of the purposes of the present invention is to provide a composition comprising mesenchymal stem cells for treating degenerative arthritis, which comprises the following components: 2×10 6 -5×10 9 cells / mL mesenchymal stem cells and 10-50ng / mL OSCAR antibody.

[0007] Preferably, the composition comprises the following components: 2×10 6 cells / mL mesenchymal stem cells and 10ng / mL OSCAR antibody.

[0008] Preferably, the composition comprises the following components: 7×10 7 cells / mL mesenchymal stem cells and 20ng / mL OSCAR antibody.

[0009] Preferably, the composition comprises the following components: 5×10 9 cells / mL mesenchymal stem cells and 50ng / mL OSCAR antibody.

[0010] Preferably, the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and / or adipose mesenchymal stem cells.

[0011] More preferably, the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells.

[0012] Preferably, the heavy chain variable region sequence of the OSCAR antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO.2.

[0013] Further preferably, the OSCAR antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5, respectively, and the OSCAR antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8, respectively.

[0014] On the other hand, the present invention also provides a method for preparing a composition comprising mesenchymal stem cells for treating degenerative arthritis, the method comprising the following steps:

[0015] 1) Isolation of mesenchymal stem cells;

[0016] 2) Preparation of OSCAR antibodies;

[0017] 3) The mesenchymal stem cells from step 1) and the OSCAR antibody from step 2) are mixed with physiological saline according to the above dosage ratio.

[0018] Preferably, step 1) separation of mesenchymal stem cells comprises the following steps:

[0019] (1) The bilateral femurs and tibias of F344 rats were taken, and the bone marrow cavity was repeatedly washed with low-glucose DMEM medium to prepare a single cell suspension;

[0020] (2) Centrifuging the cell suspension collected in step 1) to remove impurities and oil, and resuspending the cell suspension in a low-glucose DMEM medium;

[0021] (3) According to 2×10 5 -5×10 6 The cells / flask were inoculated into T75 culture flasks and placed at 37°C and 5% CO 2 Culture in an incubator and replace with fresh culture medium every 2 days;

[0022] (4) After one week of culture, the bone marrow mesenchymal stem cells were obtained by digestion with trypsin and identified by microscopic observation and flow cytometry;

[0023] (5) The identified bone marrow mesenchymal stem cells are subcultured and, after stabilization at the 3rd to 5th generation, are collected by centrifugation for later use.

[0024] Preferably, in step 2) the preparation of OSCAR antibodies is performed by using hybridoma technology to immunize BALB / c mice with OSCAR protein as an antigen, then separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells, and then screening to obtain OSCAR antibodies with higher affinity.

[0025] Further preferably, the heavy chain variable region sequence of the OSCAR antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO.2.

[0026] Further preferably, the OSCAR antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5, respectively, and the OSCAR antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8, respectively.

[0027] The advantages of the present invention are as follows: the present invention combines mesenchymal stem cells with OSCAR antibodies for the first time to treat degenerative arthritis. After verification and analysis, there is a synergistic effect between mesenchymal stem cells and OSCAR antibodies. The combination of the two can be used to inhibit the secretion of inflammatory factors such as IL-1β, TNF-ɑ and IL-6 in the subject, relieve the inflammatory damage of arthritis, and improve and promote the repair of arthritis. After administration, the symptoms of limb swelling and stiffness in the subject are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1.Analysis of serum inflammatory factor secretion before and after treatment. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0030] Example 1

[0031] A composition comprising mesenchymal stem cells for treating degenerative arthritis, comprising the following components: 2×10 6 cells / mL bone marrow mesenchymal stem cells and 10ng / mL OSCAR antibody.

[0032] The method for isolating bone marrow mesenchymal stem cells comprises the following steps:

[0033] 1) The bilateral femurs and tibias of F344 rats were obtained, and the bone marrow cavity was repeatedly washed with low-glucose DMEM medium to prepare a single cell suspension;

[0034] 2) centrifuging the cell suspension collected in step 1) to remove impurities and oil, and resuspending in low-glucose DMEM medium;

[0035] 3) Press 2×10 5 cells / flask were inoculated into T75 culture flasks and cultured in a 37°C, 5% CO2 incubator, with fresh culture medium replaced every 2 days;

[0036] 4) After one week of culture, trypsin was used for digestion to obtain bone marrow mesenchymal stem cells. After microscopic observation and flow cytometric identification, it was determined that the isolated bone marrow mesenchymal stem cells were positive for CD44, CD105, CD73 and HLA-ABC, with a positive rate of >82%; CD45, CD34 and HLA-DR were almost not expressed;

[0037] 5) The identified bone marrow mesenchymal stem cells are subcultured, and after they become stable after the fifth generation, they are collected by centrifugation for later use.

[0038] Among them, OSCAR antibody was obtained by using hybridoma technology to immunize BALB / c mice with OSCAR protein as antigen, then separating mouse spleen cells and fusing them with SP2 / 0 myeloma cells, and then screening to obtain OSCAR antibody with high affinity.

[0039] After identification, the heavy chain variable region sequence of the OSCAR antibody of the present invention is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.2. The heavy chain variable region of the OSCAR antibody includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5, respectively, and the light chain variable region of the OSCAR antibody includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8, respectively.

[0040] The preparation method of the composition comprising mesenchymal stem cells for treating degenerative arthritis comprises the following steps:

[0041] 1) Isolation of mesenchymal stem cells;

[0042] 2) Preparation of OSCAR antibodies;

[0043] 3) The mesenchymal stem cells from step 1) and the OSCAR antibody from step 2) are mixed with physiological saline according to the above dosage ratio.

[0044] Example 2

[0045] A composition comprising mesenchymal stem cells for treating degenerative arthritis, comprising the following components: 7×10 7 cells / mL bone marrow mesenchymal stem cells and 20ng / mL OSCAR antibody. The preparation method is the same as that in Example 1.

[0046] Example 3

[0047] A composition comprising mesenchymal stem cells for treating degenerative arthritis, comprising the following components: 5×10 9 cells / mL bone marrow mesenchymal stem cells and 50ng / mL OSCAR antibody. The preparation method is the same as that in Example 1.

[0048] Comparative Example 1

[0049] A composition comprising: 5×10 9 cells / mL bone marrow mesenchymal stem cells. Dissolve them in physiological saline for later use.

[0050] Comparative Example 2

[0051] A composition comprising: 50 ng / mL OSCAR antibody, which is dissolved in physiological saline for later use.

[0052] Verification Example

[0053] Preparation of rat degenerative arthritis model: The modified Hulth method was used to anesthetize F344 rats, and the skin of the hind limbs was prepared and disinfected. A longitudinal incision of about 3 cm was made on the medial side of the knee joint. The infrapatellar fold connected to the intercondylar fossa was cut to reveal the anterior cruciate ligament underneath. The anterior cruciate ligament was exposed and cut close to the femur with microscissors. The joint cavity was closed layer by layer without fixing the affected limb.

[0054] Efficacy evaluation: The rat knee arthritis model was divided into 6 groups, 4 rats in each group, wherein the control model group was treated with only saline during the whole process; the control group 1 was treated with control example 1; the control group 2 was treated with control example 2; the treatment groups 1-3 were treated with the composition described in Examples 1-3 of the present invention. A blank group was also set up, which was healthy rats and was treated with only saline during the whole process.

[0055] The dosing regimen was to inject 50 μL of sample into the rat knee joint cavity, once every 2 days. After 3 consecutive weeks of injection, the rat sample serum was collected and the secretion of IL-1β, TNF-ɑ and IL-6 in the serum was analyzed by ELISA kit.

[0056] In addition, the symptoms of the rats in Example 3 were scored before and after administration. According to the "Technical Guidelines for Clinical Trials", the swelling, stiffness and crawling ability of the rats were scored, and none, mild, moderate and severe were recorded as 0, 2, 4 and 6 points respectively. The higher the score, the more serious the condition.

[0057] The results are as follows Figure 1 As shown: Compared with the blank group, the IL-1β, TNF-ɑ and IL-6 in the serum of the rats in the control model group increased significantly, with significant differences. Compared with the control example 1-2, the IL-1β, TNF-ɑ and IL-6 in the serum of the rat model in Example 1-3 after administration and treatment decreased significantly, proving that the composition of the present invention can inhibit the secretion of inflammatory factors such as IL-1β, TNF-ɑ and IL-6, relieve the inflammatory damage of arthritis, and improve and promote the repair of arthritis.

[0058] The scoring results are shown in Table 1 below. After the administration of Example 3, the swelling, stiffness and crawling ability of the rats' limbs were significantly improved. In particular, compared with the control group, the symptoms of degenerative arthritis in the rats were significantly alleviated after treatment, and the difference was significant.

[0059] Limb swelling stiff Climbing ability Blank group 3.5±0.6 4.5±0.8 5.5±0.7 Control group 1 3.2±0.3 4.2±0.4 5.1±0.8 Control group 2 3.3±0.4 4.3±0.3 5.2±0.4 Treatment Group 3 0.8±0.2 1.2±0.4 1.6±0.8

[0060] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A composition comprising mesenchymal stem cells for treating degenerative arthritis, comprising the following components: 2×10 6 -5×10 9 cel ls / mL mesenchymal stem cells and 10-50ng / mL OSCAR antibody.

2. The composition according to claim 1, characterized in that The composition comprises the following components: 2×10 6 cel ls / mL mesenchymal stem cells and 10ng / mL OSCAR antibody.

3. The composition according to claim 1, characterized in that The composition comprises the following components: 7×10 7 cel ls / mL mesenchymal stem cells and 20ng / mL OSCAR antibody.

4. The composition according to claim 1, characterized in that The composition comprises the following components: 5×10 9 cel ls / mL mesenchymal stem cells and 50ng / mL OSCAR antibody.

5. The composition according to any one of claims 1 to 4, characterized in that The mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and / or adipose mesenchymal stem cells.

6. The composition according to claim 5, characterized in that The mesenchymal stem cells are derived from bone marrow mesenchymal stem cells.

7. The composition according to any one of claims 1 to 4, characterized in that The heavy chain variable region sequence of the OSCAR antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.

2.

8. The composition according to any one of claims 7, characterized in that The OSCAR antibody heavy chain variable region includes CDR1, CDR2 and CDR3, whose sequences correspond to SEQ ID NO.3-5, respectively; the OSCAR antibody light chain variable region includes CDR4, CDR5 and CDR6, whose sequences correspond to SEQ ID NO.6-8, respectively.

9. The method for preparing the composition for treating degenerative arthritis comprising mesenchymal stem cells according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: 1) Isolation of mesenchymal stem cells; 2) Preparation of OSCAR antibodies; 3) The mesenchymal stem cells from step 1) and the OSCAR antibody from step 2) are mixed with physiological saline according to the above dosage ratio.

10. The preparation method according to claim 9, characterized in that: Step 1), the separation of mesenchymal stem cells comprises the following steps: (1), taking bilateral femurs and tibias of F344 rats, repeatedly washing the bone marrow cavity with low-glucose DMEM medium, and preparing a single cell suspension; (2), centrifuging the cell suspension collected in step (1) to remove impurities and oil, and resuspending it in low-glucose DMEM medium; (3), at 2×10 5 -5×10 6 cells / bottle were inoculated into a T75 culture flask, placed in a 37°C, 5% CO2 incubator, and fresh culture medium was replaced every 2 days; (4) After one week of culture, the bone marrow mesenchymal stem cells were obtained by digestion with trypsin; the cells were observed under a microscope and identified by flow cytometry; (5) the identified bone marrow mesenchymal stem cells were subcultured, and after 3-5 generations of stabilization, they were collected by centrifugation for later use; Step 2) OSCAR antibody preparation is to utilize hybridoma technology to immunize BALB / c mice with OSCAR protein as antigen, isolate mouse spleen cells, fuse them with SP2 / 0 myeloma cells, and obtain OSCAR antibodies with high affinity through screening.

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