Composition containing mesenchymal stem cells for treating degenerative arthritis and preparation method thereofTechnical Field
The invention relates to the technical field of treatment of degenerative arthritis, in particular to a composition containing mesenchymal stem cells for treating the degenerative arthritis and a preparation method thereof.
Background
Degenerative arthritis is caused by the wearing of joints over time and months. This disease occurs in weight-bearing joints such as cervical vertebrae, lumbar vertebrae, knee joints, hip joints, heels, etc., and therefore long-term or excessive weight-bearing should be avoided. Cold, moisture is an important factor in pathogenesis. The disease can be caused by joints of the whole body, but is preferably caused by knee joints, hip joints, spines, finger joints and other parts with heavy loads. Especially knee and hip joint diseases.
Mesenchymal stem cells are a heterogeneous cell population derived from a matrix and can be obtained from most tissues of a human body, and a large number of experimental researches show that the mesenchymal stem cells have the differentiation potential of epidermal cells and can promote the healing of invasive skin. The marrow-derived mesenchymal stem cells have the hidden trouble of virus pollution, and the cell number and the expansion and differentiation capacity of the mesenchymal stem cells obviously decrease along with the age of a donor, so that the mesenchymal stem cells are not suitable for batch preparation. The mesenchymal stem cells derived from human umbilical cord, placenta or amniotic membrane can avoid the defects of embryo stem cell source deficiency, allograft rejection, ethics and the like, and become a good substitute for bone marrow-derived mesenchymal stem cells. The mesenchymal stem cells have self-renewal, tissue repair and immunoregulation capability, can differentiate into mesoderm lineages such as adipocytes, osteocytes, chondrocytes and the like, can differentiate into other germ lineages such as epidermal cells and vascular endothelial cells, can be easily expanded in vitro, and can keep stable differentiation capability and proliferation capability after expansion, thus being suitable for large-scale preparation.
Therefore, there is an urgent need for a safe therapeutic agent having an excellent therapeutic effect to solve the object of achieving the treatment of degenerative arthritis.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides a composition containing mesenchymal stem cells for treating degenerative arthritis and a preparation method thereof. According to the invention, the mesenchymal stem cells and the OSCAR antibody are combined for treating the degenerative arthritis for the first time, and the experimental analysis shows that the synergistic effect exists between the mesenchymal stem cells and the OSCAR antibody, and the combination of the mesenchymal stem cells and the OSCAR antibody can be used for inhibiting the secretion of inflammatory factors such as IL-1 beta, TNF-alpha, IL-6 and the like in a subject, relieving the inflammatory injury of the arthritis and improving and promoting the repair of the arthritis. And after the administration treatment, symptoms of limb swelling, stiffness and the like of the subject are obviously improved.
It is an object of the present invention to provide a composition for treating degenerative arthritis comprising mesenchymal stem cells, which comprises the following components of 2×106-5×109 cells/mL mesenchymal stem cells and 10-50ng/mLOSCAR antibody.
Preferably, the composition comprises the following components of 2X 106 cells/mL mesenchymal stem cells and 10ng/mLOSCAR antibody.
Preferably, the composition comprises 7X 107 cells/mL mesenchymal stem cells and 20ng/mL OSCAR antibody.
Preferably, the composition comprises 5X 109 cells/mL mesenchymal stem cells and 50ng/mL OSCAR antibody.
Preferably, the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and/or adipose mesenchymal stem cells.
Further preferably, the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells.
Preferably, 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.
Further preferably, the OSCAR antibody heavy chain variable region comprises CDR1, CDR2 and CDR3, the sequences of which correspond to those shown in SEQ ID nos. 3-5, respectively, and the OSCAR antibody light chain variable region comprises CDR4, CDR5 and CDR6, the sequences of which correspond to those shown in SEQ ID nos. 6-8, respectively.
In another aspect, the present invention also provides a method for preparing a composition for treating degenerative arthritis, which comprises mesenchymal stem cells, the method comprising the steps of:
1) Separating mesenchymal stem cells;
2) OSCAR antibody preparation;
3) And (3) preparing the mesenchymal stem cells in the step (1) and the OSCAR antibody in the step (2) by using normal saline according to the dosage ratio.
Preferably, the isolating of the mesenchymal stem cells of step 1) comprises the steps of:
(1) Taking femur and tibia of the two sides of the F344 rat, repeatedly washing the marrow cavity by adopting a low-sugar DMEM culture medium, and preparing single-cell suspension;
(2) Centrifuging the cell suspension collected in the step 1) to remove impurities and grease, and re-suspending the cell suspension in a low-sugar DMEM medium;
(3) Inoculating into a T75 culture flask according to2×105-5×106 cells/flask, culturing in a 37 ℃ 5% CO2 incubator, and changing fresh culture medium every 2 days;
(4) After one week of culture, adopting trypsin to make digestion treatment to obtain bone marrow mesenchymal stem cells;
(5) And carrying out subculture on the identified mesenchymal stem cells, transferring to 3-5 generations for stabilization, and centrifuging and collecting for later use.
Preferably, the preparation of the OSCAR antibody in the step 2) is to obtain the OSCAR antibody with higher affinity through screening after separating spleen cells of mice and fusing the spleen cells with SP2/0 myeloma cells after immunizing BALB/c mice with OSCAR protein as antigen by utilizing a hybridoma technology.
Further preferably, 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.
Further preferably, the OSCAR antibody heavy chain variable region comprises CDR1, CDR2 and CDR3, the sequences of which correspond to those shown in SEQ ID nos. 3-5, respectively, and the OSCAR antibody light chain variable region comprises CDR4, CDR5 and CDR6, the sequences of which correspond to those shown in SEQ ID nos. 6-8, respectively.
The invention has the advantages that the mesenchymal stem cells and the OSCAR antibody are combined for treating the degenerative arthritis for the first time, and the experimental analysis shows that the synergistic effect exists between the mesenchymal stem cells and the OSCAR antibody, and the combination of the mesenchymal stem cells and the OSCAR antibody can be used for inhibiting the secretion of inflammatory factors such as IL-1 beta, TNF-alpha, IL-6 and the like in a subject, relieving the inflammatory injury of the arthritis and improving and promoting the repair of the arthritis. And after the administration treatment, symptoms of limb swelling, stiffness and the like of the subject are obviously improved.
Drawings
FIG. 1 serum inflammatory factor secretion analysis before and after treatment.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
Example 1
A composition for treating degenerative arthritis comprising mesenchymal stem cells comprises 2×106 cells/mL bone marrow mesenchymal stem cells and 10ng/mLOSCAR antibody.
The method for separating the bone marrow mesenchymal stem cells comprises the following steps:
1) Taking femur and tibia of the bilateral sides of an F344 rat, repeatedly washing a marrow cavity by adopting a low-sugar DMEM culture medium, and preparing single-cell suspension;
2) Centrifuging the cell suspension collected in the step 1) to remove impurities and grease, and re-suspending the cell suspension in a low-sugar DMEM medium;
3) Inoculating into a T75 culture flask according to 2×105 cells/flask, culturing in a 37 ℃ and 5% CO2 incubator, and changing fresh culture medium every 2 days;
4) After one week of culture, the bone marrow mesenchymal stem cells are obtained by digestion treatment with trypsin. After microscopic observation and flow cytometry identification, determining that the isolated bone marrow mesenchymal stem cells CD44, CD105, CD73 and HLA-ABC are expressed positively with a positive rate of >82%, and CD45, CD34 and HLA-DR are hardly expressed;
5) And carrying out subculture on the identified mesenchymal stem cells, transferring to 5 generations for stabilization, and centrifuging and collecting for later use.
The OSCAR antibody is obtained by immunizing BALB/c mice with OSCAR protein as antigen by utilizing hybridoma technology, separating spleen cells of the mice, fusing the spleen cells with SP2/0 myeloma cells, and screening the spleen cells.
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. The OSCAR antibody heavy chain variable region comprises CDR1, CDR2 and CDR3, the sequences of which correspond to those shown in SEQ ID nos. 3-5, respectively, and the OSCAR antibody light chain variable region comprises CDR4, CDR5 and CDR6, the sequences of which correspond to those shown in SEQ ID nos. 6-8, respectively.
The preparation method of the composition for treating the degenerative arthritis containing the mesenchymal stem cells comprises the following steps:
1) Separating mesenchymal stem cells;
2) OSCAR antibody preparation;
3) And (3) preparing the mesenchymal stem cells in the step (1) and the OSCAR antibody in the step (2) by using normal saline according to the dosage ratio.
Example 2
A composition for treating degenerative arthritis comprising mesenchymal stem cells, which comprises the following components of 7 x 107 cells/mL bone marrow mesenchymal stem cells and 20ng/mL OSCAR antibody. The preparation method is the same as in example 1.
Example 3
A composition for treating degenerative arthritis comprising mesenchymal stem cells, which comprises the following components of 5 x 109 cells/mL bone marrow mesenchymal stem cells and 50ng/mL OSCAR antibody. The preparation method is the same as in example 1.
Comparative example 1
A composition comprising 5X 109 cells/mL of mesenchymal stem cells. Dissolving in physiological saline for use.
Comparative example 2
A composition comprising 50ng/mL OSCAR antibody. Dissolving in physiological saline for use.
Verification example
The model of degenerative arthritis was prepared by anesthetizing F344 rats using a modified Hulth method, sterilizing the hind limb skin, and making a longitudinal incision of about 3 cm on the inner side of the knee joint. The anterior cruciate ligament below the patellar fold connected with the intercondylar fossa is cut off. The anterior cruciate ligament was exposed and sheared against the femur using a micro-shear. The joint cavity is closed layer by layer, and the affected limb is not fixed.
Efficacy evaluation the rat knee arthritis model was divided into 6 groups of 4, wherein the control model group was treated with physiological saline only throughout the course, control group 1 was treated with control example 1, control group 2 was treated with control example 2, and treatment groups 1-3 were treated with the compositions of examples 1-3 of the present invention. A blank group was also set, and healthy rats were treated with normal saline alone throughout the process.
The administration scheme is that 50 mu L of samples are injected into the knee joint cavity of the rat, the samples are injected once every 2 days, serum of the rat samples is collected after 3 weeks of continuous injection, and secretion of IL-1 beta, TNF-alpha and IL-6 in the serum is analyzed by ELISA kit.
The rats of example 3 were also scored for symptoms before and after dosing, and the rats were scored for limb swelling, stiffness, and creep ability according to the guidelines of clinical trial techniques, with no, mild, moderate, and severe scores of 0,2,4, and 6, respectively, with higher scores indicating more severe disease.
The results are shown in FIG. 1, in which IL-1. Beta., TNF-. Alpha.and IL-6 were significantly increased in the serum of rats in the control model group, compared to the blank group, with significant differences. Compared with comparative examples 1-2, the composition of the invention can inhibit secretion of inflammatory factors such as IL-1 beta, TNF-alpha and IL-6, relieve inflammatory injury of arthritis and improve and promote repair of arthritis, and is proved to obviously reduce IL-1 beta, TNF-alpha and IL-6 in serum of a rat model after administration treatment of examples 1-3.
The scoring results are shown in the following table 1, and the limb swelling, stiffness and crawling ability of the rats after the administration treatment of the example 3 are obviously improved, and particularly, compared with the control group, the degenerative arthritis symptoms of the rats after the treatment are obviously relieved, and the differences are obvious.
| Swelling of limbs | Stiffness of | Crawling capability |
| 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 |
The above-described embodiments are provided to illustrate the gist of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.