Application of MybpC1 gene / protein in the preparation of drugs for osteoarthritis

Abstract

The present invention relates to the field of biomedical technologies and provides the use of the MybpC1 gene / protein in the preparation of drugs for osteoarthritis. The present invention has confirmed that the expression of the MybpC1 gene / protein is significantly down-regulated in osteoarthritis chondrocytes, and the expression levels of proteins related to the progression of osteoarthritis are related to the MybpC1 expression level. Injecting lentivirus overexpressing MybpC1 into the joint cavity of osteoarthritis mice can alleviate the progression of osteoarthritis in the mice. Therefore, the MybpC1 gene / protein can be used as a drug target and has good prospects in the screening and preparation of drugs for treating osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of MybpC1 gene / protein in the preparation of osteoarthritis drugs. Background Art

[0002] Osteoarthritis (OA) is one of the most common types of arthritis at present and belongs to a chronic degenerative disease. Its pathological features are the overall lesions of synovial joints, including the degeneration of articular cartilage, the loss of subchondral bone, the hypertrophy of synovial tissue, and the increase in the instability of tendons and ligaments, etc. Among them, the degeneration of articular cartilage is the core feature of osteoarthritis.

[0003] Currently, the treatment of osteoarthritis mainly includes drug treatment and surgical treatment, but the deficiencies of these two treatment methods are also very obvious. Drug treatment is the main way to treat osteoarthritis at present. Commonly used drugs include non-steroidal anti-inflammatory drugs (NSAIDs), adrenal cortical hormones, etc. These drugs can effectively relieve pain and inflammatory reactions and significantly improve the quality of life of patients. However, the limitation of drug treatment is that it cannot change the progression of the disease and can only relieve symptoms temporarily. Moreover, long-term use of these drugs may cause some adverse reactions and bring the risk of side effects. For severe osteoarthritis patients, surgical treatment (such as joint replacement) is a common treatment method. Surgery can effectively improve joint function, relieve pain, and improve the quality of life of patients. However, surgical treatment also has certain risks, including surgical complications, anesthesia risks, etc. In addition, the recovery period after joint replacement is relatively long, and patients need to undergo long-term rehabilitation training to restore joint function. The service life of joint replacement is usually 15 to 20 years. Therefore, young patients may need to undergo secondary or multiple surgeries. On the other hand, the cost of surgical treatment is relatively high, which may be an unaffordable burden for patients with relatively difficult economic conditions.

[0004] With the progress of molecular biology, gene therapy is expected to achieve targeted treatment of osteoarthritis, that is, by intervening in the key pathological processes of osteoarthritis, delaying or preventing the progression of osteoarthritis, and improving the quality of life of patients. Therefore, there is an urgent need to understand and identify potential biomarkers and therapeutic targets in the pathogenesis of osteoarthritis. Summary of the Invention

[0005] Aiming at the problems in the osteoarthritis treatment technology mentioned in the background art, the present invention provides the application of MybpC1 gene / protein in the preparation of osteoarthritis drugs to solve the technical problem that the existing drugs and surgeries have poor effects on the treatment of osteoarthritis.

[0006] Myosin binding protein C (MybpC) is a group of accessory proteins expressed in striated muscle, accounting for 2%-4% of the myofibrillar protein mass, including three subtypes: slow skeletal, cardiac, and fast skeletal. The molecular functions of MybpC1 (slow skeletal myosin binding protein C) include actin binding, structural molecule activation, titin binding, etc., and are involved in biological processes such as muscle contraction and cell adhesion. Mistranslation and non-translation mutations of MybpC1 may lead to severe distal joint contracture deformity and muscle tremors.

[0007] In a first aspect, the present invention provides the use of the MybpC1 gene / protein in the preparation of drugs for osteoarthritis, and the drugs include lentiviruses overexpressing MybpC1.

[0008] Furthermore, the drug treats osteoarthritis by overexpressing the MybpC1 gene.

[0009] In another aspect, the present invention provides the use of the MybpC1 gene / protein in the preparation of reagents for the auxiliary diagnosis or prognosis judgment of osteoarthritis.

[0010] Furthermore, the reagents for auxiliary diagnosis or prognosis judgment include antibodies for detecting MybpC1 protein and PCR primers for DNA strands and / or cDNA strands of the MybpC1 gene.

[0011] Furthermore, the reagent performs auxiliary diagnosis or prognosis judgment of osteoarthritis by detecting the expression level of the MybpC1 gene or protein.

[0012] The present invention has the following technical effects: Applying the MybpC1 gene / protein to the preparation of drugs for osteoarthritis can inhibit cartilage inflammatory reactions and alleviate the progression of osteoarthritis. By injecting lentiviruses overexpressing MybpC1 into the joint cavity of osteoarthritis mice, the pathological process of osteoarthritis can be intervened, and the progression of osteoarthritis in mice can be alleviated, thereby effectively treating osteoarthritis. Therefore, the MybpC1 gene / protein can be used as a drug target and has good prospects in screening and preparing drugs for treating osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] By referring to the following drawings, the exemplary embodiments of the present invention can be more fully understood:

[0014] Figure 1 This is a volcano plot showing the differential expression of MybpC1 in normal and osteoarthritis chondrocytes obtained by comparing the gene differential expression of primary chondrocytes and osteoarthritis chondrocytes in mice by RNA sequencing in Example 1 of the present invention.

[0015] Figure 2This is a picture of safranin-fast green staining and hematoxylin-eosin staining of normal and osteoarthritis clinical cartilage samples in Example 2 of the present invention; among them, A is a picture of safranin-fast green staining, scale bar: 200μm; B is a picture of hematoxylin-eosin staining, scale bar: 200μm.

[0016] Figure 3 This is to analyze the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples by immunohistochemical staining in Example 2 of the present invention. Specifically, it is a typical immunohistochemical staining picture, scale bar: 200μm.

[0017] Figure 4 This is to analyze the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples by immunohistochemical staining in Example 2 of the present invention. Specifically, it is a quantitative analysis picture of the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples, where ** indicates p < 0.01.

[0018] Figure 5 This is a picture of measuring the expression level of MybpC1 mRNA by qPCR technology (real-time fluorescence quantitative polymerase chain reaction) after inducing osteoarthritis by stimulating primary mouse chondrocytes with interleukin-1β for 48h in Example 3 of the present invention; compared with the control group, ** indicates p < 0.01.

[0019] Figure 6 This is a picture of measuring the expression level of COL II (type II collagen) mRNA by qPCR technology after inducing osteoarthritis by stimulating primary mouse chondrocytes with interleukin-1β for 48h in Example 3 of the present invention; compared with the control group, ** indicates p < 0.01.

[0020] Figure 7 This is a picture of measuring the expression level of MMP13 (matrix metalloproteinase 13) mRNA by qPCR technology after inducing osteoarthritis by stimulating primary mouse chondrocytes with interleukin-1β for 48h in Example 3 of the present invention; compared with the control group, *** indicates p < 0.001.

[0021] Figure 8 This is a picture of measuring the expression levels of MybpC1, COL II and MMP13 proteins by Western blotting immediately after stimulation and on the 2nd and 4th days after stimulation by stimulating primary mouse chondrocytes with interleukin-1β to induce osteoarthritis in Example 3 of the present invention; specifically, it is a typical picture of the expression levels of MybpC1, COL II and MMP13 proteins collected immediately after stimulation and on the 2nd and 4th days. In the study, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference.

[0022] Figure 9Quantitative analysis results of the expression level of MybpC1 protein measured by Western blotting immediately after stimulation and on the 2nd and 4th days after interleukin-1β stimulation of primary mouse chondrocytes to induce osteoarthritis in Example 3 of the present invention; compared with immediately after stimulation, * indicates p < 0.05, and ** indicates p < 0.01.

[0023] Figure 10 Quantitative analysis results of the expression level of COL II protein measured by Western blotting immediately after stimulation and on the 2nd and 4th days after interleukin-1β stimulation of primary mouse chondrocytes to induce osteoarthritis in Example 3 of the present invention; compared with immediately after stimulation, ** indicates p < 0.01, and *** indicates p < 0.001.

[0024] Figure 11 Quantitative analysis results of the expression level of MMP13 protein measured by Western blotting immediately after stimulation and on the 2nd and 4th days after interleukin-1β stimulation of primary mouse chondrocytes to induce osteoarthritis in Example 3 of the present invention; compared with immediately after stimulation, * indicates p < 0.05, and *** indicates p < 0.001.

[0025] Figure 12 Micro-CT detection results of the knee joints of mice in the sham operation group, negative control group, and experimental group in Example 4 of the present invention, specifically typical pictures of micro-CT of the knee joint bone tissue of the three groups.

[0026] Figure 13 Statistical analysis pictures of the bone volume fraction of the knee joints of mice in the sham operation group, negative control group, and experimental group in Example 4 of the present invention; compared with the experimental group, * indicates p < 0.05.

[0027] Figure 14 Statistical analysis pictures of the bone mineral density of the knee joints of mice in the sham operation group, negative control group, and experimental group in Example 4 of the present invention; compared with the experimental group, * indicates p < 0.05.

[0028] Figure 15 Typical pictures of hematoxylin-eosin staining, safranin-fast green staining, and toluidine blue staining of knee joint tissue sections of mice in the sham operation group, negative control group, and experimental group in Example 4 of the present invention, scale bar: 200 μm.

[0029] Figure 16 Statistical analysis pictures of the Osteoarthritis Research Society International (OARSI) score of knee joint tissue sections of mice in the sham operation group, negative control group, and experimental group in Example 4 of the present invention; ** indicates p < 0.01, and *** indicates p < 0.001.

[0030] Figure 17This is the immunohistochemical staining picture of the knee joint tissue sections of the sham operation group, negative control group and experimental group of mice in Example 4 of the present invention. Specifically, it is the typical picture of immunohistochemistry of COL II, MMP13 and MybpC1 in the three groups, scale bar: 200μm.

[0031] Figure 18 This is the statistical analysis picture of the immunohistochemical results of COL II in the knee joint tissue of the sham operation group, negative control group and experimental group of mice in Example 4 of the present invention; *** indicates p < 0.001.

[0032] Figure 19 This is the statistical analysis picture of the immunohistochemical results of MMP13 in the knee joint tissue of the sham operation group, negative control group and experimental group of mice in Example 4 of the present invention; ** indicates p < 0.01.

[0033] Figure 20 This is the statistical analysis picture of the immunohistochemical results of MybpC1 in the knee joint tissue of the sham operation group, negative control group and experimental group of mice in Example 4 of the present invention; ** indicates p < 0.01, *** indicates p < 0.001. Detailed implementation manners

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0036] In some embodiments, the present invention provides the application of MybpC1 gene / protein in the preparation of drugs for osteoarthritis, and the drugs include lentivirus with overexpression of MybpC1.

[0037] Specifically, the drug treats osteoarthritis by overexpressing the MybpC1 gene.

[0038] In some embodiments, the present invention provides the application of MybpC1 gene / protein in the preparation of reagents for the auxiliary diagnosis or prognosis judgment of osteoarthritis.

[0039] Specifically, the reagents for auxiliary diagnosis or prognosis judgment include antibodies for detecting MybpC1 protein or PCR primers for DNA strands and / or cDNA strands of MybpC1 gene.

[0040] Specifically, the reagent is used for the auxiliary diagnosis or prognosis judgment of osteoarthritis by detecting the expression level of the MybpC1 gene or protein.

[0041] Experimental reagents:

[0042] The total RNA extraction kit, reverse transcription reagent kit, and SYBR reagent (synergistic binding reagent) were purchased from Takara.

[0043] The forward and reverse primers of the genes of MMP13 (matrix metalloproteinase 13), COL II (type II collagen), MybpC1 (slow skeletal myosin-binding protein C), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were all purchased from Shanghai Sangon Biotech Co., Ltd., and the sequences are shown in Table 1:

[0044] Table 1 Sequences of forward and reverse primers of MMP13, COL II, MybpC1, and GAPDH genes

[0045]

[0046] Example 1: Comparison of differential gene expression between primary mouse chondrocytes and osteoarthritic chondrocytes by RNA sequencing

[0047] Interleukin-1β is one of the most commonly used inflammatory cytokines to induce inflammation in chondrocytes. In this example, interleukin-1β was used to treat primary mouse chondrocytes to induce the transformation of chondrocytes into the osteoarthritic state, and then RNA sequencing comparative analysis was performed on normal primary mouse chondrocytes and osteoarthritic chondrocytes.

[0048] As Figure 1 shown, the gene detection results were presented as a volcano plot, and the analysis results showed that the expression level of the MybpC1 gene was significantly decreased in osteoarthritic chondrocytes, suggesting that the MybpC1 gene may play an important biological role in the occurrence and development of osteoarthritis.

[0049] Example 2: Further demonstration that the MybpC1 gene / protein affects the occurrence and development of osteoarthritis through clinical samples

[0050] Ten joint cartilage samples from healthy knees that needed to be amputated due to trauma or malignant tumors were collected as normal cartilage samples, and ten joint cartilage samples from osteoarthritis patients who needed knee replacement surgery were collected as clinical osteoarthritic cartilage samples. All 20 patients with cartilage samples provided detailed clinical information and signed informed consent forms, and the ethical review batch number was: IIT2024425.

[0051] As Figure 2As shown, safranin-fast green staining and hematoxylin-eosin staining were performed on normal and osteoarthritis clinical cartilage samples, and it can be seen that the articular cartilage of osteoarthritis patients has suffered severe damage.

[0052] As Figure 3 and Figure 4 shown, immunohistochemical staining was used to analyze the expression levels of MybpC1 protein in normal and osteoarthritis clinical cartilage samples. It can be seen that the expression level of MybpC1 protein in osteoarthritis clinical cartilage samples decreased, further proving that MybpC1 protein plays a key role in the occurrence and development of osteoarthritis.

[0053] Example 3: Inducing osteoarthritis in primary mouse chondrocytes by interleukin-1β

[0054] (1) Primary mouse chondrocytes were collected and cultured, divided into two groups: a control group and an experimental group. The control group was not treated with anything, and the experimental group was stimulated with 10 ng / ml of interleukin-1β to induce osteoarthritis in primary mouse chondrocytes. After 48 hours, RNA was collected, and qPCR technology (real-time fluorescence quantitative polymerase chain reaction) was used to detect the expression levels of the mRNAs of MybpC1, COL II, and MMP13.

[0055] As Figures 5 - 7 shown, it can be seen that after stimulation with interleukin-1β, primary mouse chondrocytes produced inflammation, the expression level of the inflammation-related gene MMP13 gene increased significantly, and the expression level of the COL II gene with articular cartilage protective effect decreased, indicating that interleukin-1β successfully induced osteoarthritis in primary mouse chondrocytes. At the same time, it can be seen that the expression level of the MybpC1 gene in chondrocytes after osteoarthritis occurred decreased.

[0056] (2) Primary mouse chondrocytes were collected and cultured, divided into two groups: a control group and an experimental group. The control group was not treated with anything, and the experimental group was stimulated with 10 ng / ml of interleukin-1β to induce osteoarthritis in chondrocytes. Cell proteins were collected immediately after stimulation and on the 2nd and 4th days, and the expression levels of MybpC1, COL II, and MMP13 proteins were detected by Western blotting.

[0057] As Figures 8 - 11 shown, it can be seen that after stimulation with interleukin-1β, chondrocytes produced inflammation, the expression level of the inflammation-related protein MMP13 protein increased significantly, while the expression level of the COL II protein with articular cartilage protective effect decreased significantly. As the stimulation time increased, the expression level of MybpC1 protein gradually decreased.

[0058] Example 4: Construction of a mouse model of osteoarthritis to further demonstrate the effect of MybpC1 gene / protein expression on the progression of osteoarthritis

[0059] (1)Construction of destabilization of medial meniscus (DMM): By cutting the tibial ligament of the medial meniscus of the mouse and freeing the medial meniscus, the knee joint of the mouse was made unstable. During the movement of the mouse, the injury of the medial cartilage of the mouse was aggravated, resulting in osteoarthritis in the mouse. In the sham operation group, only the joint cavity was opened, and the medial meniscus was not freed; in the negative control group, empty lentivirus was injected into the joint cavity of the mice with the medial meniscus instability model; in the experimental group, lentivirus overexpressing MybpC1 was injected into the joint cavity of the mice with the medial meniscus instability model. The changes of osteoarthritis in the knee joint of the mouse were observed by micro-CT examination. The ethical review batch number of this animal experiment was: DM20210816.

[0060] As Figures 12 - 14 shown, the micro-CT examination results showed that the tibial plateau in the negative control group was severely damaged and osteophyte hyperplasia was obvious, which was in line with the manifestations of osteoarthritis. After injecting lentivirus overexpressing MybpC1, the damage of the tibial plateau was reduced and the osteophytes were relatively fewer.

[0061] (2)Sections of the knee joint tissues of the mice in the sham operation group, negative control group and experimental group were made and stained with hematoxylin and eosin, safranin-fast green and toluidine blue respectively to observe the protective effect of MybpC1 protein on cartilage in the mouse body.

[0062] As Figure 15 and Figure 16 shown, the cartilage surface of the mice in the sham operation group was intact and no obvious osteophytes were formed; while the cartilage degeneration in the negative control group was severe, with more osteophytes formed, and it could be seen from the Osteoarthritis Research Society International score that the score of the negative control group was significantly higher than that of the sham operation group; while the degeneration of the knee joint cartilage in the experimental group was reduced and the osteophytes were decreased, and it could be seen from the Osteoarthritis Research Society International score that the score of the experimental group was significantly lower than that of the negative control group.

[0063] (3) Sections of the knee joint tissues of the mice in the sham operation group, negative control group and experimental group were made and immunohistochemically stained.

[0064] As Figures 17 - 20As shown, the expression level of MybpC1 in the negative control group was significantly lower than that in the sham operation group, and the expression level of MybpC1 in the experimental group was significantly higher than that in the negative control group; the expression levels of COL II and MMP13 in the negative control group were significantly decreased and increased respectively compared with the sham operation group; after transfection with lentivirus overexpressing MybpC1, compared with the negative control group, the downward trend of COL II and the upward trend of MMP13 in the experimental group were significantly improved. In summary, it shows that intra-articular injection of lentivirus overexpressing MybpC1 can alleviate the progression of osteoarthritis in mice by inhibiting cartilage inflammatory response and promoting collagen formation.

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of MybpC1 gene / protein in the preparation of osteoarthritis drugs, characterized in that: The drug includes a MybpC1-overexpressing lentivirus.

2. The use according to claim 1, characterized in that: The drug treats osteoarthritis by overexpressing the MybpC1 gene.

3. Application of reagents for detecting MybpC1 gene / protein in the preparation of reagents for auxiliary diagnosis of osteoarthritis.

4. The use according to claim 3, characterized in that: The auxiliary diagnosis reagents include antibodies for detecting MybpC1 protein or PCR primers for detecting the cDNA chain of the MybpC1 gene.

5. The use according to claim 4, characterized in that: The auxiliary diagnosis reagent performs auxiliary diagnosis of osteoarthritis by detecting the expression level of MybpC1 gene or protein.

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