Application of MybpC1 gene / protein in preparation of osteoarthritis drugs

Abstract

The invention relates to the technical field of biological medicines, and provides application of MybpC1 gene / protein in preparation of osteoarthritis medicines. The invention proves that the expression of the MybpC1 gene / protein in osteoarthritis cartilage cells is obviously reduced, the expression level of related protein of osteoarthritis progress is related to the expression level of MybpC1, and the progress of osteoarthritis of a mouse can be relieved by injecting MybpC1 overexpressed lentivirus into an articular cavity of the mouse with osteoarthritis. Therefore, the MybpC1 gene / protein can be used as a drug target, and has a good prospect in screening and preparing drugs for treating osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular 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 and is a chronic degenerative disease. Its pathological characteristics are the overall lesions of synovial joints, including degeneration of articular cartilage, loss of subchondral bone, hypertrophy of synovial tissue, and increased instability of tendons and ligaments, among which degeneration of articular cartilage is the core feature of osteoarthritis.

[0003] At present, the treatment of osteoarthritis mainly includes drug therapy and surgical treatment, but the shortcomings of these two treatment methods are also very obvious. Drug therapy is currently the main way to treat osteoarthritis. Commonly used drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), adrenocortical 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 therapy is that it cannot change the progression of the disease and can only temporarily relieve symptoms. Moreover, long-term use of these drugs may cause some adverse reactions and bring side effects. For patients with severe osteoarthritis, 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 and anesthesia risks. In addition, the recovery period after joint replacement is long, and patients need long-term rehabilitation training to restore joint function. The service life of joint replacement is usually 15 to 20 years, so young patients may need to undergo secondary or multiple surgeries. On the other hand, the cost of surgical treatment is high, which may be an unbearable burden for patients with more difficult economic conditions.

[0004] With the progress of molecular biology, gene therapy is expected to achieve targeted treatment of osteoarthritis, that is, to delay or prevent the progression of osteoarthritis and improve the quality of life of patients by intervening in the key pathological processes of osteoarthritis. Therefore, it is urgent to understand and identify potential biomarkers and therapeutic targets in the pathogenesis of osteoarthritis. Summary of the invention

[0005] In view of the problems in osteoarthritis treatment technology mentioned in the background technology, the present invention provides the use of MybpC1 gene / protein in the preparation of osteoarthritis drugs to solve the technical problem that drugs and surgeries in the prior art are not effective in treating osteoarthritis.

[0006] Myosin Binding Protein C (MybpC) is a group of auxiliary proteins expressed in skeletal muscle, accounting for 2%-4% of the mass of myofibril protein, 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 it participates in biological processes such as muscle contraction and cell adhesion. Mistranslation and untranslated mutations of MybpC1 may lead to severe distal joint flexion deformity and muscle tremor.

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

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

[0009] On the other hand, the present invention provides the use of MybpC1 gene / protein in preparing a reagent for auxiliary diagnosis or prognosis of osteoarthritis.

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

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

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

[0013] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 This is a volcano plot of the differential expression of MybpC1 in normal and osteoarthritic chondrocytes obtained by comparing the differential expression of genes between mouse primary chondrocytes and osteoarthritic chondrocytes through RNA sequencing in Example 1 of the present invention.

[0014] Figure 2These are pictures of safranin fast green staining and hematoxylin and eosin staining of normal and osteoarthritis clinical cartilage samples according to Example 2 of the present invention; A is a picture of safranin fast green staining, scale bar: 200 μm; B is a picture of hematoxylin and eosin staining, scale bar: 200 μm.

[0015] Figure 3 In Example 2 of the present invention, the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples was analyzed by immunohistochemical staining, specifically a typical immunohistochemical staining picture, scale bar: 200 μm.

[0016] Figure 4 This is Example 2 of the present invention, in which the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples was analyzed by immunohistochemical staining, specifically, a quantitative analysis picture of the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples, wherein ** indicates p<0.01.

[0017] Figure 5 This is a picture of Example 3 of the present invention, in which osteoarthritis was induced by stimulating primary mouse chondrocytes with interleukin-1β, and the expression level of MybpC1 mRNA was measured by qPCR technology (real-time fluorescence quantitative polymerase chain reaction) 48 hours later; compared with the control group, ** indicates p < 0.01.

[0018] Figure 6 This is a picture of Example 3 of the present invention, in which osteoarthritis was induced by stimulating primary mouse chondrocytes with interleukin-1β, and the expression level of COL II (type II collagen) mRNA was measured by qPCR technology 48 hours later; compared with the control group, ** indicates p < 0.01.

[0019] Figure 7 This is a picture of Example 3 of the present invention, in which osteoarthritis was induced by stimulating primary mouse chondrocytes with interleukin-1β, and the expression level of MMP13 (matrix metalloproteinase 13) mRNA was determined by qPCR technology 48 hours later; compared with the control group, *** indicates p < 0.001.

[0020] Figure 8 This is a picture of Example 3 of the present invention, in which osteoarthritis is induced by stimulating mouse primary chondrocytes with interleukin-1β, and the expression levels of MybpC1, COL II and MMP13 proteins are measured by western blotting immediately after stimulation and after 2 and 4 days; specifically, it is a typical picture of the expression levels of MybpC1, COL II and MMP13 proteins collected immediately after stimulation and after 2 and 4 days. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference in the study.

[0021] Fig. 9This is the result of quantitative analysis of Example 3 of the present invention in which osteoarthritis was induced by stimulating mouse primary chondrocytes with interleukin-1β, and the expression level of MybpC1 protein was determined by Western blotting immediately after stimulation and 2 and 4 days later; compared with that immediately after stimulation, * indicates p < 0.05, and ** indicates p < 0.01.

[0022] Fig.10 This is the result of quantitative analysis of Example 3 of the present invention in which osteoarthritis was induced by stimulating mouse primary chondrocytes with interleukin-1β, and the expression level of COL II protein was determined by Western blotting immediately after stimulation and 2 and 4 days later; compared with that immediately after stimulation, ** indicates p < 0.01, and *** indicates p < 0.001.

[0023] Fig.11 This is the result of quantitative analysis of Example 3 of the present invention, in which osteoarthritis was induced by stimulating mouse primary chondrocytes with interleukin-1β, and the expression level of MMP13 protein was determined by Western blotting immediately after stimulation and 2 and 4 days later; compared with that immediately after stimulation, * indicates p < 0.05, and *** indicates p < 0.001.

[0024] Fig.12 These are the micro-CT test 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 images of micro-CT of the knee joint bone tissues of the three groups.

[0025] Fig.13 This is a statistical analysis picture of the knee joint bone volume fraction 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.

[0026] Fig.14 This is a statistical analysis picture of the knee joint bone density 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] Fig.15 These are typical pictures of hematoxylin and 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.

[0028] Fig.16 This is a statistical analysis picture of the International Osteoarthritis Research Society scores of the 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, *** indicates p < 0.001.

[0029] Fig.17These are immunohistochemical staining images 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, specifically typical images of COL II, MMP13 and MybpC1 immunohistochemistry of the three groups, scale bar: 200 μm.

[0030] Fig.18 This is a statistical analysis picture of the COL II immunohistochemistry results of the knee joint tissues of mice in the sham operation group, negative control group and experimental group in Example 4 of the present invention; *** indicates p<0.001.

[0031] Fig.19 This is a statistical analysis picture of the MMP13 immunohistochemistry results of the knee joint tissues of mice in the sham operation group, negative control group and experimental group in Example 4 of the present invention; ** indicates p<0.01.

[0032] Fig. 20 This is a statistical analysis picture of the MybpC1 immunohistochemistry results of the knee joint tissues of mice in the sham operation group, negative control group and experimental group in Example 4 of the present invention; ** indicates p < 0.01, *** indicates p < 0.001. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 intended to limit the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0035] In some embodiments, the present invention provides the use of MybpC1 gene / protein in the preparation of osteoarthritis drugs, and the drugs include MybpC1 overexpressing lentivirus.

[0036] Specifically, the drug treats osteoarthritis by overexpressing the MybpC1 gene.

[0037] In some embodiments, the present invention provides the use of the MybpC1 gene / protein in the preparation of reagents for auxiliary diagnosis or prognosis of osteoarthritis.

[0038] Specifically, the reagents for auxiliary diagnosis or prognosis judgment include antibodies for detecting MybpC1 protein or PCR primers for the DNA chain and / or cDNA chain of the MybpC1 gene.

[0039] Specifically, the reagent performs auxiliary diagnosis or prognosis of osteoarthritis by detecting the expression level of MybpC1 gene or protein.

[0040] Experimental reagents: Total RNA extraction kit, reverse transcription reagent kit and SYBR reagent (synergistic binding reagent) were purchased from Takara.

[0041] The primers for MMP13 (matrix metalloproteinase 13), COL II (type II collagen), MybpC1 (slow skeletal myosin binding protein C), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) genes were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The sequences are shown in Table 1: Table 1 Sequences of primers before and after MMP13, COL II, MybpC1 and GAPDH genes

[0042] Example 1: Comparison of differential gene expression between mouse primary chondrocytes and osteoarthritis chondrocytes by RNA sequencing 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 chondrocytes to transform into an osteoarthritis state, and then RNA sequencing was performed to compare and analyze normal mouse primary chondrocytes and osteoarthritis chondrocytes.

[0043] like Figure 1 As shown, the genetic test results were presented as a volcano plot. The analysis results showed that the expression level of MybpC1 gene in osteoarthritis chondrocytes was significantly reduced, suggesting that MybpC1 gene may play an important biological role in the occurrence and development of osteoarthritis.

[0044] Example 2: Further evidence from clinical samples that MybpC1 gene / protein affects the occurrence and development of osteoarthritis Articular cartilage samples from 10 healthy knees that required amputation due to trauma or malignant tumors were collected as normal cartilage samples, and articular cartilage samples from 10 osteoarthritis patients who required knee replacement surgery were collected as clinical cartilage samples for osteoarthritis. All patients with the 20 cartilage samples provided detailed clinical data and signed informed consent forms. The ethical review approval number is: IIT2024425.

[0045] like Figure 2 As shown, safranin fast green staining and hematoxylin eosin staining of normal and osteoarthritis clinical cartilage samples showed that the articular cartilage of osteoarthritis patients suffered serious damage.

[0046] like Figure 3 and Figure 4 As shown, the expression level of MybpC1 protein in normal and osteoarthritis clinical cartilage samples was analyzed by immunohistochemical staining. It can be seen that the expression level of MybpC1 protein in osteoarthritis clinical cartilage samples was reduced, further proving that MybpC1 protein plays a key role in the occurrence and development of osteoarthritis.

[0047] Example 3: Induction of osteoarthritis by stimulating mouse primary chondrocytes with interleukin-1β (1) Mouse primary chondrocytes were collected and cultured and divided into a control group and an experimental group. The control group did not receive any treatment, while the experimental group was stimulated with 10 ng / ml interleukin-1β to induce osteoarthritis. RNA was collected 48 hours later, and the expression levels of MybpC1, COL II, and MMP13 mRNA were detected using qPCR (real-time fluorescence quantitative polymerase chain reaction) technology.

[0048] like Figure 5-Figure 7 As shown, it can be seen that after stimulation by interleukin-1β, mouse primary chondrocytes produce inflammation, the expression level of the inflammation-related gene MMP13 gene is significantly increased, and the expression level of the COL II gene with articular cartilage protection effect is reduced, indicating that interleukin-1β successfully induces osteoarthritis in mouse primary chondrocytes. At the same time, it can be seen that the expression level of the MybpC1 gene in chondrocytes after osteoarthritis is reduced.

[0049] (2) Mouse primary chondrocytes were collected and cultured and divided into a control group and an experimental group. The control group did not receive any treatment, while the experimental group was supplemented with 10 ng / ml interleukin-1β to stimulate chondrocytes to induce osteoarthritis. 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.

[0050] like Figure 8-Figure 11 As shown, it can be seen that after stimulation by interleukin-1β, chondrocytes produce inflammation, the expression level of the inflammation-related protein MMP13 protein is significantly increased, while the expression level of the COL II protein with articular cartilage protection effect is significantly decreased. As the stimulation time increases, the expression level of the MybpC1 protein gradually decreases.

[0051] Example 4: Construction of an osteoarthritis mouse model to further demonstrate the effect of MybpC1 gene / protein expression on the progression of osteoarthritis (1) Construction of Destabilization of Medial Meniscus (DMM): The medial meniscus tibial ligament of the mice was cut and the medial meniscus was freed, causing instability in the mice's knee joints. During the movement of the mice, the damage to the medial cartilage of the mice was aggravated, causing osteoarthritis in the mice. In the sham operation group, only the joint cavity was opened without freeing the medial meniscus; in the negative control group, empty lentivirus was injected into the joint cavity of the mice with medial meniscus destabilization model; in the experimental group, lentivirus overexpressing MybpC1 was injected into the joint cavity of the mice with medial meniscus destabilization model. The changes in osteoarthritis of the mice's knee joints were observed by micro-CT examination. The ethical review approval number of this animal experiment is: DM20210816.

[0052] like Figure 12-14 As shown in the figure, the micro-CT examination results showed that the tibial plateau in the negative control group was severely damaged and osteophytes were obvious, which was consistent with the manifestation of osteoarthritis. After the injection of the lentivirus overexpressing MybpC1, the tibial plateau damage was alleviated and osteophytes were relatively few.

[0053] (2) The knee joint tissues of the mice in the sham operation group, negative control group, and experimental group were sectioned and stained with hematoxylin and eosin, safranin fast green, and toluidine blue to observe the protective effect of MybpC1 protein on cartilage in mice.

[0054] like Fig.15 and Fig.16 As shown, the cartilage surface of mice in the sham operation group was intact, and no obvious osteophyte formation was observed; while the cartilage of the negative control group was severely degenerated, with more osteophytes formed, and the score of the negative control group was significantly higher than that of the sham operation group, as shown in the International Osteoarthritis Research Society scoring; while the knee cartilage degeneration in the experimental group was alleviated, osteophytes were reduced, and the score of the experimental group was significantly lower than that of the negative control group, as shown in the International Osteoarthritis Research Society scoring.

[0055] (3) The knee joint tissues of mice in the sham operation group, negative control group, and experimental group were sectioned and immunohistochemically stained.

[0056] like Figure 17-Figure 20 As shown in the figure, the expression of MybpC1 in the negative control group was significantly lower than that in the sham operation group, and the expression of MybpC1 in the experimental group was significantly higher than that in the negative control group; the expression of COL II and MMP13 in the negative control group was significantly decreased and increased compared with the sham operation group; after MybpC1 was transfected by lentivirus, the decreasing trend of COL II and the increasing trend of MMP13 in the experimental group were significantly improved compared with the negative control group. In summary, the knee joint cavity injection of MybpC1 overexpressing lentivirus can alleviate the progression of osteoarthritis in mice by inhibiting cartilage inflammatory response and promoting collagen formation.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should 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 MybpC1 gene / protein in the preparation of reagents for auxiliary diagnosis or prognosis of osteoarthritis.

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

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

Images