Application of integrin alpha4 in preparation of rheumatoid arthritis detection kit and medicine

Abstract

The invention relates to the technical field of biological medicines, and provides application of integrin alpha4 in preparation of rheumatoid arthritis detection kits and drugs, an integrin alpha4 inhibitor can down-regulate the expression or activity of integrin alpha4 at a gene level and / or a protein level, can hinder activation of part of downstream molecules, and can inhibit the expression or activity of the integrin alpha4 at the gene level and / or the protein level. The inflammatory reaction before rheumatoid arthritis is inhibited, and the effect of improving and treating rheumatoid arthritis is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of integrin α4 in the preparation of rheumatoid arthritis detection kits and drugs. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects the joints. In my country, there are 2.8 to 7 million new cases of RA each year. RA cannot be cured and requires long-term effective drug intervention. However, common anti-rheumatic drugs (DMARDs) used in clinical practice currently have primary or secondary drug resistance. Some patients cannot receive effective treatment, which causes various chronic clinical symptoms and seriously affects their quality of life.

[0003] Therefore, we proposed integrin α4 as an intervention target for rheumatoid arthritis and the application of integrin α4 in the preparation of rheumatoid arthritis detection kits and drugs. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides the use of integrin α4 in the preparation of a rheumatoid arthritis detection kit and a drug.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] Application of a reagent for detecting the expression level of integrin α4 or its encoding gene in a detection kit for rheumatoid arthritis.

[0009] Use of integrin α4 inhibitors in the preparation of drugs for treating rheumatoid arthritis.

[0010] Furthermore, the drug for treating rheumatoid arthritis is a drug for treating rheumatoid arthritis in humans, mice, cats, dogs, cattle or sheep.

[0011] Furthermore, the integrin α4 inhibitor can regulate the expression or activity of integrin α4 at the gene level and / or protein level.

[0012] Furthermore, the integrin α4 inhibitor is selected from an integrin α4 specific antibody or a small molecule inhibitor of the integrin α4 encoding gene.

[0013] Furthermore, the small molecule inhibitor of the integrin α4 encoding gene is siRNA, miRNA, dsRNA or shRNA.

[0014] Among them, the sense strand of siRNA is 5′-GCCAUAGUCUCUCUCUUAUTT-3′ (SEQ ID NO. 1);

[0015] Antisense strand 5'-AUAAGAGAGAGACUAΜGGCTT-3' (SEQ ID NO. 2).

[0016] (III) Beneficial effects

[0017] The present invention provides the use of integrin α4 in the preparation of a rheumatoid arthritis detection kit and a drug, which has the following beneficial effects:

[0018] 1. The study found that the regulatory network where ITGA4 is located is mainly enriched in the extracellular matrix receptor pathway (ECM-receptor interaction), the adhesion factor pathway (FAK, focal adhesion kinase) and the PI3K-AKT signaling pathway. ITGA4 is located in the core position of the three signaling pathways and can be associated with upstream and downstream pathways, forming a regulatory mechanism with ITGA4 on the cell membrane surface as the key node;

[0019] 2. Studies have shown that during the onset of rheumatoid arthritis, inflammatory factors in the body attract the extracellular matrix (ECM) to accumulate in the affected joints and bind to ITGA4 as a ligand on the surface of synovial cells. The activated ITGA4 recruits intracellular adhesion factors (FAK), which in turn activates downstream active molecules such as PI3K and AKT, forming an ECM-ITGA4-FAK-PI3K-AKT signal transduction pathway, promoting the proliferation of synovial cells in rheumatoid arthritis and the secretion of inflammatory factors, thereby forming pathological hyperplasia of joint tissue and persistent inflammatory response, triggering symptoms of rheumatoid arthritis;

[0020] 3. Studies have found that compared with osteoarthritis, ITGA4 is expressed at a high level in clinical samples of rheumatoid arthritis patients, making it a diagnostic target for distinguishing osteoarthritis from rheumatoid arthritis; using siRNA technology to specifically interfere with ITGA4 expression will cause the activation level of some downstream proteins in the regulatory pathway, hinder the activation of some downstream molecules, relieve the patient's persistent inflammatory response, and reduce the symptoms of rheumatoid arthritis caused by inflammatory response, which has an improvement and therapeutic effect on rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a bioinformatics analysis of synovial cells in rheumatoid arthritis; A is the heat map and volcano map of differentially expressed genes screened by the chip; B is the GSEA gene function analysis; C is the differentially expressed gene network interaction analysis; and D is the key hub genes determined by four algorithms.

[0022] Figure 2 This is an analysis of the signal pathway of the regulatory network in which ITGA4 is located; A is the signal pathway in which ITGA4 participates in two synovial cells; B is the expression of genes in the regulatory network in which ITGA4 is located in the ECM, FAK and PI3K-AKT signal pathways; C is the ECM, FAK and PI3K-AKT 33 pathways are associated with ITGA4 to form the ECM-ITGA4-FAK-PI3K-AKT regulatory mechanism.

[0023] Figure 3 This is an analysis of ITGA4 at the cellular level, where A is the flow cytometry sorting of rheumatoid arthritis synovial fibroblasts and synovial macrophages; B is the expression comparison of ITGA4 in osteoarthritis synovial cells and rheumatoid arthritis synovial cells, ITGA4 is expressed at a significantly high level in rheumatoid arthritis synovial cells; C is an immunoblot detection, after siRNA interferes with the expression of ITGA4 in synovial cells, its downstream molecules hinder the activation of some downstream molecules.

[0024] Figure 4 This is the immunohistochemical analysis of ITGA4 animal experiments; A is the comparison of the contents of three inflammatory factors, and B is the comparison of the pathological changes in the ankle joint tissues of mice corresponding to the three inflammatory factors shown by different staining. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] 1. Study the main mechanism of ITGA4 regulating the occurrence of rheumatoid arthritis through synovial cell chip analysis

[0028] The GEO database was used to screen the expression profile chip of rheumatoid arthritis synovial cells, and the common osteoarthritis synovial cells were used as the control for subsequent bioinformatics analysis. After screening, the synovial fibroblast expression profile chip GSE56409 and the synovial macrophage expression profile chip GSE100786 met the requirements. The online GEO2R analysis tool was used to perform differential expression analysis of all genes, with P value <0.05 and |log2 fold change (FC)|>1 as the critical values ​​for screening differentially expressed genes (DEGs). GSEA and KEGG were used for GO and KEGG pathway enrichment analysis, with P adjusted value <0.05 as the cutoff value. Protein interaction analysis and visualization were performed using the STRING database and Cytoscape 3.7.1, with a confidence value >0.4 as the cutoff standard. The results are shown in Figure 1 As shown in the results, ITGA4 is a common key node gene in synovial fibroblasts and synovial macrophages, and is expressed at a high level in osteoarthritis. Gene function analysis shows that ITGA4 is mainly involved in biological processes such as immune regulation, immune cell differentiation, and cell signal transduction.

[0029] Further analysis of the signal pathway of the regulatory network of ITGA4 was performed, and the results were as follows Figure 2 As shown, ITGA4 is mainly enriched in the extracellular matrix receptor pathway (ECM-receptor interaction), adhesion factor pathway (focaladhesion) and PI3K-AKT signaling pathway. It plays a positive regulatory role in all three signaling pathways and integrates its pathways with cell membrane receptors to form an ECM-ITGA4-FAK-PI3K-AKT regulatory network with ITGA4 as the core regulator.

[0030] 2. Verification of ITGA4-mediated downstream signaling pathways using synoviocytes

[0031] (1) Primary culture of rheumatoid arthritis synovial cells

[0032] The primary culture of synovial cells adopts the common tissue block culture method. The simple steps of culture are as follows: ask the nurse to put the specimen into low-temperature physiological saline (stationary) on the operating table. When the operation is completed, put the specimen into PBS ice water and store it in a sterilized container. The tissue is cut open and the white and smooth synovial tissue at the joint fold and hyperplasia is carefully identified. The hyperplastic pannus attached to the articular cartilage surface is also synovial tissue. The fat tissue is carefully removed and the red blood cells are removed and then minced sharply with dissecting scissors. Digest with 4mg / ml collagenase at double the volume of the obtained tissue and incubate at 37℃ for 120 minutes, then replace with trypsin to continue digestion for 30 minutes. The cell suspension is filtered through a 70um cell filter and immediately cultured for primary synovial cells. The medium is changed every 2-3 days. RA synovial cells mainly include synovial fibroblasts and synovial macrophages. Before the synovial cells are passaged for 3 times, some synovial macrophages will be mixed in the synovial cells. At this time, flow cytometry can be used to separate the two types of cells. After the synovial cells are passaged for 5-7 times, due to the lack of nutrients necessary for the growth of macrophages, the macrophages in the synovial cells will gradually be transferred to the synovial fibroblasts, which account for more than 95%. They can be stably passaged and cultured for subsequent experiments.

[0033] (2) siRNA regulation of gene expression

[0034] Primary rheumatoid arthritis synovial cells were used for siRNA knockdown experiments. ITGA4 siRNA or negative control siRNA was diluted in Lipofectamine RNAiMAX and Opti-MEM serum medium, 10 μl Lipofectamine RNAiMAX transfection reagent was added to 250 μl Opti-MEM serum medium, and 10 μl siRNA was added to 250 μl Opti-MEM serum medium, and primary cultured pericytes were transfected according to the manufacturer's protocol. After 24 hours, the transfection medium was removed, and the transfected cells were cultured in a medium with low serum concentration for 24 hours. After that, the cell culture supernatant (conditioned medium) was collected, and the cells were lysed to obtain RNA, and the knockdown effect of ITGA4 mRNA was verified by qPCR. The same culture medium and the negative control (scramble) of the above-mentioned transfected siRNA were used for pericyte transfection as a negative control.

[0035] ITGA4 siRNA:

[0036] Sense strand 5′-GCCAUAGUCUCUCUCUUAUTT-3′ (SEQ ID NO. 1);

[0037] Antisense strand 5'-AUAAGAGAGAGACUAΜGGCTT-3' (SEQ ID NO. 2).

[0038] Negative control siRNA:

[0039] Sense strand 5'-UUCUCCGAACGΜGUCACGUTT-3' (SEQ ID NO. 3);

[0040] Antisense strand 5'-ACGΜGACACGUUCGGAGAATT-3' (SEQ ID NO. 4).

[0041] In this example, when ITGA4 siRNA and negative control siRNA were synthesized, two Ts at the ends replaced U, which did not affect the inhibitory effect, reduced costs, and increased the ability to resist enzymatic hydrolysis.

[0042] The low-serum medium and primary culture medium both used RPMI1640 medium (Hyclone). The low-serum medium contained 1% FBS (fetal bovine serum), and the primary culture medium contained 10% FBS. The culture conditions were 37°C, 5% CO 2 .

[0043] TNF-α was used to stimulate cells to simulate a high inflammatory factor environment, making the phenotype of primary synovial cells of rheumatoid arthritis closer to the true characteristics of rheumatoid arthritis. Total cell protein was extracted and Western blotting was used to detect ITGA4 and downstream protein expression.

[0044] Instructions for TNF-α stimulation of cells: When the cell culture density is about 80%, discard the original culture medium and replace with serum-free culture medium (RPMI1640) for 12 hours. Then add TNF-α (novoprotein) to a final concentration of 5ug / ml and stimulate for 12 hours. After the stimulation is complete, discard the culture medium containing TNF-α and replace with regular culture medium (containing 10% FBS) for continued culture for subsequent experiments.

[0045] The results are as follows Figure 3 As shown, ITGA4 is indeed expressed at a high level in rheumatoid arthritis synovial cells, and is involved in regulating the activation of downstream molecules in related pathways, specifically downregulating the expression level of ITGA4 in synovial primary cultured cells; after siRNA interferes with the expression of ITGA4 in synovial cells, it can hinder the expression and activation of downstream molecules FAK, PI3K, and AKT, reduce the secretion of inflammatory factors in rheumatoid arthritis, slow down the persistent inflammatory response in patients, and alleviate the symptoms of rheumatoid arthritis caused by inflammatory response.

[0046] 3. Use animal models to verify the effects of ITGA4 on rheumatoid arthritis symptoms and related inflammatory responses

[0047] Establishment of animal model of rheumatoid arthritis

[0048] The animal model of rheumatoid arthritis was established by inducing C57BL / 6 mice with type II collagen booster immunization. C57BL / 6 wild-type mice and ITGA4 knockout mice (C57BL / 6JCya-Itga4em1flox / Cya conditional knockout mice, purchased from Saiye (Suzhou) Biotechnology Co., Ltd.) were selected for RA modeling. C57BL mice have inflammatory response characteristics and a high modeling rate. The specific modeling steps are as follows: 20 C57BL mice (20±5g, 6-8 weeks) were used to construct a collagen-induced arthritis model by the CIA method, and the RA model was established by booster immunization. The first collagen injection was on the first day of the experiment, and the mice were subcutaneously injected with an equal volume of collagen (chicken type II collagen, 2mg / ml) and Freund's complete adjuvant totaling 100ul. The booster immunization was 28 days after the first injection, and the mice were subcutaneously injected with an equal volume of collagen and Freund's incomplete adjuvant totaling 100ul. After the first immunization, the mice's joints were observed every 7 days for inflammatory changes, and the changes were judged based on joint redness, swelling and movement disorders. After the booster immunization, the mice were observed and judged every 3 days until the 56th day after the start of the experiment (behavioral judgment indicated that the model was successful). The success rate of the RA model in C57BL mice without any intervention by the booster immunization method was 70%-80%.

[0049] Gene knockout mouse CIA model group: ITGA4 gene knockout C57BL / 6 mouse type II collagen rheumatoid arthritis model;

[0050] Wild-type C57BL / 6 mouse model group: wild-type C57BL / 6 mouse type II collagen rheumatoid arthritis model;

[0051] Blank control group: The experimental mice were immunized synchronously with physiological saline instead of type II collagen.

[0052] The serum and spleen cells of the established rheumatoid arthritis mouse model were collected, and the pro-inflammatory factors (IL-1β / IL-6 / TNF-α) in the serum and spleen cell culture supernatant were detected.

[0053] Serum separation and acquisition instructions: After the mouse was anesthetized with ether, about 1 mL of blood was collected from the mouse heart and placed in a 1.5 mL centrifuge tube. The tube was placed at 4°C for 4 hours and centrifuged at 12000 rpm at 4°C for 10 minutes. The upper serum was collected for inflammatory factor detection.

[0054] Instructions for isolation and culture of spleen cells: After mice were anesthetized with ether, the spleen was isolated and washed with PBS three times. Single spleen cells were ground and classified with a 100-mesh sieve. Red blood cells were lysed with 0.17 M ammonium chloride and then washed three times with RPMI1640 culture medium. The cells were counted with a cell counting plate. 1×10 spleen cells were collected from each group. 6 The spleen cells were cultured at 37°C and 5% CO 2The culture medium was RPMI1640 complete medium (10% FBS, 1% P / S). The upper culture medium was collected after culturing spleen cells for 48 hours for quantitative analysis of spleen cytokines TNF-α, IL-1β, and IL-6.

[0055] The inflammatory status of ITGA4 gene knockout C57BL mice induced with RA and wild-type C57BL mice induced with RA is shown in the following table. Figure 4 As shown, after ITGA4 gene knockout, IL-1β, TNF-α, and IL-6 were significantly reduced, and the inflammatory response of the knockout mouse CIA model group was significantly lower than that of the wild-type C57BL / 6 mouse model group, and was closer to the blank control group. (In Figure A, WT is the wild-type C57BL / 6 mouse model group, KO is the knockout mouse CIA model group, and in Figure B, normal is the blank control group, model is the wild-type C57BL / 6 mouse model group, and siRNA is the knockout mouse CIA model group.)

[0056] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "includes..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a reagent for detecting the expression level of integrin α4 or its encoding gene in a detection kit for rheumatoid arthritis.

2. Application of integrin α4 inhibitors in the preparation of drugs for the treatment of rheumatoid arthritis.

3. Use of the integrin α4 inhibitor according to claim 2 in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The drug for treating rheumatoid arthritis is a drug for treating rheumatoid arthritis in humans, mice, cats, dogs, cattle or sheep.

4. Use of the integrin α4 inhibitor according to claim 2 or 3 in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The integrin α4 inhibitor can regulate the expression or activity of integrin α4 at the gene level and / or protein level.

5. Use of the integrin α4 inhibitor according to claim 4 in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The integrin α4 inhibitor is selected from an integrin α4 specific antibody or a small molecule inhibitor of the integrin α4 encoding gene.

6. Use of the integrin α4 inhibitor according to claim 5 in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The small molecule inhibitor of the integrin α4 encoding gene is siRNA, miRNA, dsRNA or shRNA.

7. Use of the integrin α4 inhibitor according to claim 6 in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The sense strand of siRNA was 5′-GCCAUAGUCUCUCUCUUAUTT-3′; Antisense strand 5′-AUAAGAGAGAGACUAΜGGCTT-3′.

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