A composition for treating degenerative arthritis containing mesenchymal stem cells and a preparation method thereof

Abstract

The present invention relates to a composition for treating degenerative arthritis containing mesenchymal stem cells and a preparation method thereof. The present invention for the first time combines mesenchymal stem cells with OSCAR antibody for treating degenerative arthritis. Through verification and analysis, there is a synergistic effect between mesenchymal stem cells and OSCAR antibody, and the combination of the two can be used to inhibit the secretion of inflammatory factors such as IL-1β, TNF-α and IL-6 in a subject, relieve the inflammatory damage of arthritis, and can improve and promote the repair of arthritis. Moreover, after administration treatment, symptoms such as limb swelling and stiffness of the subject are significantly improved.

Description

Technical Field

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

[0002] Degenerative arthritis is caused by years of wear and tear of joints. This disease mostly occurs in weight-bearing joints, such as the cervical vertebra, lumbar vertebra, both knees, hips, heels, etc., so long-term or excessive weight-bearing should be avoided. Cold and humidity are important pathogenic factors. This disease can occur in all joints of the body, but it is prone to occur in weight-bearing joints such as the knee joint, hip joint, spine and finger joints. Especially the knee and hip joint lesions are more common.

[0003] Mesenchymal stem cells are a heterogeneous cell population derived from the stroma and 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 for epidermal cell differentiation and can promote the healing of damaged skin. Bone marrow-derived mesenchymal stem cells are not suitable for batch preparation because of the risk of virus contamination and the obvious decline in cell quantity, amplification and differentiation ability with the increase of donor age. Mesenchymal stem cells derived from human umbilical cord, placenta or amniotic membrane can avoid many limitations such as the lack of embryonic stem cell sources, allogeneic rejection, and ethical issues, and have become good substitutes for bone marrow-derived mesenchymal stem cells. Mesenchymal stem cells have the abilities of self-renewal, tissue repair and immune regulation, and can differentiate into mesodermal lineage cells, such as adipocytes, osteocytes and chondrocytes, etc. 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 amplify in vitro, and their differentiation ability and proliferation ability remain stable after amplification, which is suitable for large-scale preparation.

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

[0005] Aiming at the technical problems existing in the prior art, the present invention provides a composition for treating degenerative arthritis containing mesenchymal stem cells and a preparation method thereof. The present invention first combines mesenchymal stem cells with OSCAR antibodies for the treatment of degenerative arthritis. Through verification and analysis, there is a synergistic effect between mesenchymal stem cells and OSCAR antibodies. The two can be combined to inhibit the secretion of inflammatory factors such as IL-1β, TNF-α and IL-6 in the body of a subject, relieve the inflammatory damage of arthritis, and can improve and promote the repair of arthritis. And after drug administration treatment, the symptoms such as limb swelling and stiffness of the subject are significantly improved.

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

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

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

[0009] Preferably, the composition comprises the following components: 5×10 9 cells / mL of mesenchymal stem cells and 50 ng / mL of 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 as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2.

[0013] More preferably, the heavy chain variable region of the OSCAR antibody comprises CDR1, CDR2, and CDR3, and their sequences correspond to those shown in SEQ ID NOs.3 - 5 respectively. The light chain variable region of the OSCAR antibody comprises CDR4, CDR5, and CDR6, and their sequences correspond to those shown in SEQ ID NOs.6 - 8 respectively.

[0014] On the other hand, the present invention also provides a preparation method of a composition for treating degenerative arthritis containing mesenchymal stem cells, and the method comprises the following steps:

[0015] 1) Isolation of mesenchymal stem cells;

[0016] 2) Preparation of OSCAR antibody;

[0017] 3) Mix the mesenchymal stem cells in step 1) with the OSCAR antibody in step 2) according to the dosage ratio and configure with physiological saline.

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

[0019] (1). Take the bilateral femurs and tibias of F344 rats, and repeatedly wash the bone marrow cavities with low-glucose DMEM medium to prepare a single-cell suspension.

[0020] (2). Centrifuge the cell suspension collected in step (1) to remove impurities and grease, and resuspend it in low-glucose DMEM medium.

[0021] (3). Inoculate at 2×10 5 -5×10 6 cells / bottle into a T75 culture flask, and culture it in an incubator at 37°C and 5% CO2. Replace the fresh medium every 2 days.

[0022] (4). After culturing for one week, treat with trypsin to obtain bone marrow mesenchymal stem cells; observe under a microscope and identify by flow cytometry.

[0023] (5). Passage culture the identified bone marrow mesenchymal stem cells. After reaching stability at passages 3-5, centrifuge and collect for standby.

[0024] Preferably, the preparation of the OSCAR antibody in step (2) is to immunize BALB / c mice with OSCAR protein as an antigen using the hybridoma technology, then isolate the mouse spleen cells and fuse them with SP2 / 0 myeloma cells, and finally obtain the OSCAR antibody with high affinity through screening.

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

[0026] More preferably, the heavy chain variable region of the OSCAR antibody includes CDR1, CDR2, and CDR3, and their sequences correspond to those shown in SEQ ID NOs.3-5 respectively. The light chain variable region of the OSCAR antibody includes CDR4, CDR5, and CDR6, and their sequences correspond to those shown in SEQ ID NOs.6-8 respectively.

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

[0028] Figure 1 . Analysis of the secretion of serum inflammatory factors before and after treatment. Detailed implementation manners

[0029] The present invention will be 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 for treating degenerative arthritis containing mesenchymal stem cells, which comprises the following components: 2×10 6 cells / mL bone marrow mesenchymal stem cells and 10 ng / mL OSCAR antibody.

[0032] Among them, the method for isolating bone marrow mesenchymal stem cells includes the following steps:

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

[0034] 2) Centrifuge the cell suspension collected in step 1) to remove impurities and grease, and resuspend it in low-glucose DMEM medium;

[0035] 3) Inoculate into a T75 culture flask at 2×10 5 cells / flask, and culture in an incubator at 37°C and 5% CO2. Replace the fresh medium every 2 days;

[0036] 4) After culturing for one week, digest with trypsin to obtain bone marrow mesenchymal stem cells. After microscopic observation and flow cytometry identification, it is determined that the isolated bone marrow mesenchymal stem cells are positive for the expression of CD44, CD105, CD73 and HLA-ABC, and the positive rate > 82%; CD45, CD34 and HLA-DR are hardly expressed;

[0037] 5) The identified bone marrow mesenchymal stem cells are subcultured. After being stable at passage 5, centrifuge and collect for standby.

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

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

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

[0041] 1) Isolation of mesenchymal stem cells;

[0042] 2) Preparation of OSCAR antibody;

[0043] 3) Mix the mesenchymal stem cells in step 1) and the OSCAR antibody in step 2) according to the dosage ratio and configure with normal saline.

[0044] Example 2

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

[0046] Example 3

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

[0048] Control Example 1

[0049] A composition, which includes: 5×10 9 cells / mL bone marrow mesenchymal stem cells. Dissolve it in normal saline for standby.

[0050] Control Example 2

[0051] A composition, which includes: 50 ng / mL OSCAR antibody. Dissolve it in normal saline for standby.

[0052] Verification Example

[0053] Preparation of a rat model of degenerative arthritis: Using the modified Hulth method, anesthetize F344 rats, prepare the skin of the hind limbs and disinfect, and make a longitudinal incision about 3 cm long on the medial side of the knee joint. Cut the infrapatellar fold connected to the intercondylar fossa, and the anterior cruciate ligament below it can be seen. Expose the anterior cruciate ligament and cut the anterior cruciate ligament close to the femur with micro scissors. Close the joint cavity layer by layer without fixing the affected limb.

[0054] Efficacy evaluation: Divide the rat knee arthritis model into 6 groups, with 4 rats in each group. Among them, in the control model group, only normal saline is used to treat the rat model throughout the process; in control group 1, the rat model is treated with control example 1; in control group 2, the rat model is treated with control example 2; treatment groups 1-3 are treated with the compositions described in Examples 1-3 of the present invention to treat the rat model. Another blank group is set, which is healthy rats and only treated with normal saline throughout the process.

[0055] The dosing regimen is to inject 50 μL of the sample into the knee joint cavity of the rats, inject once every 2 days, and after continuous injection for 3 weeks, collect the serum of the rat samples and analyze the secretion of IL-1β, TNF-α and IL-6 in the serum through an ELISA kit.

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

[0057] The results are as Figure 1 shown: Compared with the blank group, the levels of IL-1β, TNF-α and IL-6 in the serum of the rats in the control model group were significantly increased, showing significant differences. Compared with control examples 1-2, after treatment with the compositions of Examples 1-3, the levels of IL-1β, TNF-α and IL-6 in the serum of the rat models decreased significantly, proving that the compositions of the present invention can inhibit the secretion of inflammatory factors such as IL-1β, TNF-α and IL-6, relieve the inflammatory damage of arthritis, and can improve and promote the repair of arthritis.

[0058] The scoring results are shown in Table 1 below. After treatment with Example 3, the limb swelling, stiffness and crawling ability of the rats were significantly improved. Especially compared with the control group, the symptoms of degenerative arthritis in the rats were significantly relieved after treatment, and the differences were significant.

[0059] Limb swelling Stiffness Crawling 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 functions of the above embodiments are to illustrate the substantial content of the present invention, but do not limit the protection scope of the present invention thereby. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. A composition for treating degenerative arthritis containing mesenchymal stem cells, which comprises the following components: 2×10 6 -5×10 9 cells / mL of mesenchymal stem cells and 10 - 50 ng / mL of OSCAR antibody, wherein the heavy chain variable region of the OSCAR antibody comprises CDR-H1, CDR-H2 and CDR-H3, and their sequences respectively correspond to those shown in SEQ ID NO.3 - 5, and the light chain variable region of the OSCAR antibody comprises CDR-L1, CDR-L22 and CDR-L3, and their sequences respectively correspond to those shown in SEQ ID NO.6 - 8.

2. The composition according to claim 1, wherein The composition comprises the following components: 2×10 6 mesenchymal stem cells per mL and 10 ng / mL OSCAR antibody.

3. The composition according to claim 1, wherein The composition comprises the following components: 7×10 7 mesenchymal stem cells per mL and 20 ng / mL of OSCAR antibody.

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

5. The composition according to any one of claims 1-4, 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, wherein 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 as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO.2.

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