Construction method and application of rheumatoid arthritis combined atherosclerosis disease model

Abstract

The invention relates to the technical field of disease animal model construction methods, and provides a construction method of a rheumatoid arthritis and atherosclerosis disease model, which is characterized in that an hTNF-a transgenic mouse and an APOE transgenic mouse are used for mating and breeding, and the hTNF-a and APOE transgenic mice are screened out for modeling. The modeling method provided by the invention can be close to a model of human rheumatoid arthritis combined with cardiovascular diseases, and modeling is simple and practical.

Description

Technical Field

[0001] This invention relates to the technical field of animal model construction methods for diseases, specifically to a method for constructing and applying a disease model of rheumatoid arthritis combined with atherosclerosis. Background Technology

[0002] Rheumatoid arthritis (RA), an autoimmune disease characterized by chronic synovial inflammation, has a significantly higher incidence of cardiovascular disease in its patients than in the general population. RA combined with cardiovascular disease is also a leading cause of death in RA patients. From a clinical perspective, this model can simulate the pathophysiological state of coexisting with these two diseases, providing an ideal research platform for revealing the potential mechanisms underlying the predisposition to atherosclerosis in RA patients. The persistent inflammatory response, immune dysregulation, and metabolic abnormalities in RA patients may accelerate the development of atherosclerosis through multiple pathways, while traditional single-disease models struggle to fully reflect this complex interaction. By constructing a combined model, the effects of RA-related inflammatory factors (such as TNF-α and IL-6) on vascular endothelial function, lipid metabolism, and plaque formation can be observed more directly, thus providing experimental evidence to elucidate the association between the two diseases.

[0003] hTNFα transgenic mice are one of the commonly used animal models for studying rheumatoid arthritis (RA). Their core characteristic is the use of genetic engineering to continuously express human tumor necrosis factor α (hTNFα) in mice, thus spontaneously producing a pathological phenotype similar to human RA. hTNFα transgenic mice typically begin to show joint lesions at a few weeks of age, initially manifesting as synovitis. As the disease progresses, joint swelling and deformities gradually develop, and in severe cases, multiple joints (such as the ankle and knee joints) can be affected. The lesions are chronic and progressive, highly similar to the course of human RA. Furthermore, this model can also be accompanied by systemic inflammatory responses, such as elevated levels of serum inflammatory factors (IL-6, IL-1, etc.), and some mice may exhibit mild damage to other organs.

[0004] APOE transgenic disease mice are important animal models for studying lipid metabolism disorders and atherosclerosis. Their core technology involves modifying the mouse apolipoprotein E (APOE) gene using genetic engineering to mimic the pathophysiological processes of related human diseases. Regarding pathological characteristics, APOE... - / -Mice exhibit significant lipid metabolism disorders even on a normal diet, characterized by markedly elevated plasma total cholesterol and low-density lipoprotein cholesterol levels. With age, they spontaneously form atherosclerotic plaques, primarily affecting the aortic root and aortic arch. Later stages may involve plaque rupture and thrombosis, mirroring the progression of human atherosclerosis. This model provides an ideal framework for studying the association between human APOE gene polymorphisms and disease. To better simulate the pathogenesis of RA combined with atherosclerosis in humans and to screen for effective targeted therapies, there is an urgent need to construct an animal model of RA combined with atherosclerosis.

[0005] Based on this, the present invention provides a method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis.

[0007] On the one hand, the present invention provides a method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis, which involves mating and breeding hTNF-a transgenic mice and APOE transgenic mice, and screening out hTNF-a and APOE transgenic mice for modeling.

[0008] Furthermore, the selected hTNF-a and APOE transgenic mice were fed a Western diet for at least 10 weeks.

[0009] Furthermore, the nutritional composition of the Western diet is as follows: it contains 17.3% protein, 21.2% fat and 48.5% carbohydrates, of which the fat includes 12.8% saturated fat, 5.6% monounsaturated fat and 1% polyunsaturated fat.

[0010] Furthermore, during the Western diet, transgenic mice underwent weekly orbital blood sampling, weight measurement, and joint thickness measurement.

[0011] Furthermore, transgenic mice fed the Western diet for at least 10 weeks were sacrificed and their aortic and ankle tissues were extracted.

[0012] Furthermore, the serum of transgenic mice fed the Western diet for at least 10 weeks was tested weekly for total cholesterol, triglycerides, and low-density lipoprotein cholesterol.

[0013] Furthermore, after extracting the mouse aorta, the aorta was frozen sectioned, stained with hematoxylin and eosin (HE) and Oil Red O, and the size of the aortic plaque was detected.

[0014] Furthermore, after extracting ankle joint tissue, the mouse ankle joint was subjected to Micro-CT examination, followed by decalcification and paraffin embedding, and sectioning for HE and Fast Green staining.

[0015] On the other hand, the rheumatoid arthritis combined with atherosclerosis disease model constructed by this method can be used to study the disease mechanism of rheumatoid arthritis and / or atherosclerosis.

[0016] On the other hand, the rheumatoid arthritis combined with atherosclerosis disease model constructed by this method can be used to screen drugs for the treatment of rheumatoid arthritis and / or atherosclerosis.

[0017] The beneficial effects of this invention are as follows: This invention constructs a model by crossbreeding and screening hTNF-α transgenic mice with APOE transgenic mice, combined with a specific Western diet. This comprehensive approach can more fully and accurately simulate the complex pathophysiological process of rheumatoid arthritis combined with atherosclerosis in humans. hTNF-α plays a key pro-inflammatory role in the pathogenesis of rheumatoid arthritis, the APOE gene is closely related to the formation of atherosclerosis, and the Western diet, rich in high-fat and high-cholesterol components, is an important factor in inducing cardiovascular disease and aggravating the inflammatory response of rheumatoid arthritis in humans. Therefore, this model simulates the human disease environment from both the genetic and dietary induction levels. Compared with traditional single-factor induced models, it can more realistically reflect the mechanism of disease occurrence and development in the human body, providing a more reliable experimental subject for in-depth research on the nature of the disease.

[0018] The modeling process of this invention is rationally designed, with clear and specific operating steps, and has high reproducibility. From the mating, breeding, and screening of transgenic mice, to the feeding of Western diets, and then to the determination of various detection indicators, each step has detailed and standardized operating methods. It does not require complex and expensive equipment and technology, and general laboratory conditions can meet the experimental requirements.

[0019] The disease model constructed in this invention exhibits good stability. Under the same experimental conditions, repeated experiments yielded relatively consistent results, with high similarity in disease manifestations, physiological indicators, and histopathological changes in mice. This stability provides a solid foundation for subsequent large-scale drug screening and disease mechanism research, ensuring the reliability and scientific rigor of experimental data and contributing to accurate and credible research conclusions. Attached Figure Description

[0020] Figure 1The images show frozen sections of mouse aortic arch stained with HE and Oil Red O; tissue sections of the aortic arch from healthy control mice, Apoe- and hTNF-α / APOE- mice stained with HE (top) and Oil Red O (bottom), scale bar = 100 μm; Figure 2 Serum cholesterol, triglyceride, and low-density lipoprotein concentrations in mice; serum cholesterol (A), triglyceride (B), and low-density lipoprotein concentrations (C) in healthy control mice, Apoe- and hTNF-α / APOE- mice; Figure 3 Joint thickness and arthritis incidence index in mice, (A) Arthritis incidence index in healthy control mice, hTNF-α and hTNF-α / APOE mice, (B) Joint thickness was monitored every 7 days; Figure 4 Micro-CT 3D reconstruction of mouse ankle joint; Figure 5 HE and O staining of mouse ankle joints. HE staining (top) and O staining (bottom) of healthy control mice, hTNF-α mice, and hTNF-α / APOE mice. Figure 6 Mouse gene identification: Electrophoresis diagram of gene identification in Apoe- and hTNF-α transgenic mice. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This invention provides a method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis. hTNF-a transgenic mice and APOE transgenic mice are crossbred to screen out hTNF-a and APOE transgenic mice (hereinafter referred to as TA transgenic mice).

[0023] Furthermore, 8-week-old TA transgenic mice were fed a Western diet and a normal diet (experimental control group) for 10 weeks. During the experiment, the joint thickness of the TA transgenic mice was measured weekly. After 10 weeks of feeding, the mice were sacrificed and blood, hind leg ankle joints, and aortas were collected.

[0024] The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis includes the following technical solutions: (1) Screening out hTNF-a and APOE transgenic mice (TA transgenic mice for short); (2) The AT mouse genotype was identified by PCR technology, and AT transgenic mice were screened and fed a Western diet for 10 weeks. (3) During the Western diet, blood was collected from the orbital cavity weekly, and the weight and joint thickness of the mice were measured. (4) Mice were sacrificed after 10 weeks on a Western diet, and tissues from the aorta and ankle joints were extracted. (5) The total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the serum of mice were measured weekly; (6) Frozen sections of the aorta were prepared, stained with hematoxylin and eosin (HE) and Oil Red O, and the size of the aortic plaques was determined. (7) Micro-CT was performed on the ankle joint of the mouse. Then, the ankle joint was decalcified and embedded in paraffin, and the sections were stained with HE and O-fast green.

[0025] The following specific examples will provide further details.

[0026] Example 1 In an SPF-grade animal facility, hTNF-α transgenic mice (Cyagen Biotech Co., Ltd.) and APOE-- mice (Jicui Biotechnology Co., Ltd.) were bred together. Their offspring were then genotyped and screened to identify transgenic mice containing only hTNF-α, transgenic mice containing APOE--, and transgenic mice containing both hTNF-α and APOE--.

[0027] Mice were housed in an SPF-grade environment with a constant temperature of 20-26℃, humidity controlled at 40%-70%, and a 12-hour light-dark cycle.

[0028] The nutritional composition of the Western diet is as follows: 17.3% protein, 21.2% fat (12.8% saturated fat, 5.6% monounsaturated fat, and 1% polyunsaturated fat) and 48.5% carbohydrates. All feed is purchased from the Guangdong Provincial Experimental Animal Center.

[0029] A normal diet consists of 25% protein, 14% fat, and 60% carbohydrates.

[0030] Six 8-week-old wild-type mice were used as the control group, while six 8-week-old hTNF-α transgenic mice, six 8-week-old APOE-transgenic mice, and six 8-week-old hTNF-α / APOE-transgenic mice were fed a Western diet for 10 weeks. Body weight and joint thickness were recorded weekly during the feeding process.

[0031] After 10 weeks of Western dietary intervention, serum samples from each group were collected for quantitative analysis of triglycerides (TG), total cholesterol, and low-density lipoprotein. Various aortas were extracted and stained with Oil Red O. Frozen sections of the aortic arch were then subjected to hematoxylin / eosin (HE) staining and Oil Red O (ORO) staining to analyze aortic plaque. Various mouse ankle joints were extracted for 3D bone tissue reconstruction using Micro-CT. Subsequently, the ankle joints of each group were decalcified and paraffin-embedded sections, and HE and ORO staining was performed to analyze bone destruction and joint integrity.

[0032] Detection method: Gene identification: Sample collection and preprocessing, fresh samples from the tip of mouse tails were selected for direct DNA extraction; Genomic DNA extraction: Digest tissue with lysis buffer (containing proteinase K) and incubate at 55°C until completely dissolved; purify DNA using a kit or the phenol-chloroform method; PCR amplification reaction system preparation (operate on ice): Mix 2×PCR Master Mix, upstream and downstream specific primers, genomic DNA template and enzyme-free water according to the proportions in the table below.

[0033]

[0034] The hTNF-α primers are: R: 5'-TTAGG GTCGGAACCCAAGCTTTG-3'; F: 5'-GCACTGACCTCCCACTTTCCCT-3', with a positive sequence of 380 bp.

[0035] The APOE mouse primers are: R: 5'-TGCCTAGTCTCGGCTGAACTAC; F: 3'-CAACCTGGGCTACACTAATTGAC, with a positive sequence of 346 bp.

[0036] Agarose gel electrophoresis detection: Prepare 2% agarose gel (containing nucleic acid dye), calculate and weigh: 2% = 2 g agarose powder / 100 mL 1×TAE buffer.

[0037] Weigh 2.0 g of electrophoresis-grade agarose into a clean 250 mL Erlenmeyer flask and add 100 mL of 1×TAE. Heat the gel: Cover the flask opening with plastic wrap and poke 3 small holes. Microwave on high for 30 seconds, remove and shake well, then microwave for another 30 seconds. Repeat twice until the solution is clear, free of particles, and just boiling. Add water and weigh: Weigh the total weight after dissolving the gel. Use hot ddH2O to replenish the evaporated amount to ensure accurate concentration. Cool and add dye: Let stand at room temperature for 3 minutes, then add nucleic acid dye: EB: 0.5 µg / mL when it is no longer hot to the touch (≈ 55℃). Gently shake to mix, avoiding air bubbles. Pour the gel and insert the comb: Seal both ends of the gel casting plate and place it horizontally; insert a 1 mm thick comb, with the lower edge of the teeth 1 mm from the bottom of the plate. Slowly pour the gel solution along the flask opening to a thickness of 4 mm; if air bubbles appear, remove them with a pipette tip while still hot. Solidify: Let stand at room temperature for 30 minutes. The gel should be uniformly milky white and transparent; a "fog" at the edges indicates it has solidified. Remove the comb vertically. Electrophoresis: Transfer the gel plate to the electrophoresis tank, add the same batch of 1×TAE buffer, ensuring the buffer level is approximately 1 mm above the gel surface; then load the samples. Load the PCR products and DNA marker, and electrophores at 110V for 25 minutes; observe the bands using a gel imaging system. Select mice that are positive for both hTNF-α and APOE for a Western diet experiment.

[0038] The Arthritis Index (AII) is used to assess the progression of joint inflammation. The AII scores are as follows: 0: Normal joint; 1: Swelling and redness in 1-2 interphalangeal joints; 2: Affecting 3-4 interphalangeal joints or 1 large joint; 3: Swelling and redness in more than 4 joints; 4: Arthritis affecting the entire foot / paw. Each rat's bilateral hind limbs can be scored from 0 to 8 using this system.

[0039] Serum triglycerides (TG), total cholesterol, and low-density lipoprotein (LDL) assays: Triglycerides (TG) and total cholesterol were measured according to the Beyotime kit instructions. Low-density lipoprotein (LDL) was measured according to the kit instructions provided by Solarbio.

[0040] Aortic and ankle joint examination: Micro-CT scans were performed on the joints of each group of animals to construct 3D models and assess changes in joint structure. Scanning parameters included a resolution of 18 mm, a voltage of 60 kV, a current of 400 μA, and a 0.1 mm filter. Reconstruction and analysis were performed automatically. All image data were analyzed and reconstructed using specialized software. The score for radiation damage to the joint bones was from 0 to 3: 0, no damage; 1, mild; 2, moderate; 3, severe.

[0041] HE staining: Routine dewaxing and rehydration. Staining: Place sections in hematoxylin-eosin staining solution for 5-10 minutes. Wash with water: Wash several times with distilled water to remove the stain on the surface. Routine rehydration and xylene clearing; mount with optical resin. Examine and photograph under a microscope.

[0042] Safranin O staining of cartilage: Routine dewaxing and rehydration; staining with freshly prepared iron-hematoxylin staining solution for 3-5 minutes, followed by rinsing with PBS for 3 minutes; differentiation with acidic differentiation solution for 15 seconds; washing with distilled water for 10 minutes. Staining in Fast Green solution for 3-5 minutes; rapid washing of the sections with a weak acid solution for 10-15 seconds to remove nonspecific Fast Green; staining the sections in Safranin O solution for 5 minutes; dehydration with 95% ethanol and anhydrous ethanol; clearing with xylene; mounting with optical resin; and microscopic examination and photography.

[0043] Oil Red O staining: Place fresh frozen sections of the aorta at room temperature for 2 minutes. Specimen fixation: Fix the sample with 4% paraformaldehyde, ensuring sufficient concentration and complete fixation. Staining: Immerse the sections in the prepared Oil Red O staining solution and stain at room temperature for 30 minutes to 2 hours. Staining solution preparation: Oil Red O can be dissolved in a mixture of 50% ethanol and 10% salicylic acid to prepare a 0.5% Oil Red O solution. Washing: Quickly rinse the stained sections with 60% isopropanol or 70% ethanol to remove excess staining solution. Counterstaining: Counterstain the sections with 10% Mayer hematoxylin to stain the cell nuclei blue. Mounting: Mount with glycerol gelatin, dry, and observe under a microscope.

[0044] Figure 1 The images show frozen sections of mouse aortic arch stained with HE and Oil Red O; tissue sections of the aortic arch from healthy control mice, Apoe- and hTNF-α / APOE- mice stained with HE (top) and Oil Red O (bottom), scale bar = 100 μm. Figure 1 Apoe- and hTNF-α / APOE- mice showed a significant increase in aortic arch plaques compared to healthy controls.

[0045] Figure 2 Serum cholesterol, triglyceride, and low-density lipoprotein concentrations in mice; serum cholesterol (A), triglyceride (B), and low-density lipoprotein concentrations (C) in healthy control mice, Apoe- and hTNF-α / APOE- mice. Figure 2 Compared with the healthy control group, Apoe- and hTNF-α / APOE- mice showed significantly increased serum cholesterol, triglyceride and low-density lipoprotein concentrations.

[0046] Figure 3Joint thickness and arthritis incidence index in mice, (A) Arthritis incidence index in healthy control mice, hTNF-α and hTNF-α / APOE mice, (B) Joint thickness was monitored every 7 days; Figure 3 The study showed that hTNF-α and hTNF-α / APOE-- mice had significantly higher arthritis incidence and joint thickness than the healthy control group.

[0047] Figure 4 Micro-CT 3D reconstruction of mouse ankle joint; Figure 4 The study showed that ankle joint damage was significantly increased in hTNF-α and hTNF-α / APOE-- mice compared to healthy control mice.

[0048] Figure 5 HE and O staining of mouse ankle joints. HE staining (top) and O staining (bottom) of healthy control mice, hTNF-α mice, and hTNF-α / APOE mice. Figure 5 The study showed severe damage to the ankle cartilage and joints in hTNF-α and hTNF-α / APOE mice. These results indicate that hTNF-α / APOE mice exhibit symptoms of both rheumatoid arthritis-related joint destruction and arterial plaque formation.

[0049] Figure 6 Gene identification in mice: Electrophoresis images of Apoe- and hTNF-α transgenic mice. Gene identification screened mice that were positive for both Apoe- and hTNF-α.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis, characterized in that, hTNF-a transgenic mice were crossbred with APOE transgenic mice to screen for hTNF-a and APOE transgenic mice for modeling.

2. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to claim 1, characterized in that, The selected hTNF-a and APOE transgenic mice were fed a Western diet for at least 10 weeks.

3. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to claim 2, characterized in that, The nutritional composition of the Western diet is as follows: it contains 17.3% protein, 21.2% fat and 48.5% carbohydrates, of which the fat includes 12.8% saturated fat, 5.6% monounsaturated fat and 1% polyunsaturated fat.

4. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to claim 2, characterized in that, During the Western diet, transgenic mice underwent weekly blood sampling from their orbits, and their weight and joint thickness were measured.

5. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to claim 2, characterized in that, Transgenic mice fed the Western diet for at least 10 weeks were sacrificed and their aortic and ankle tissues were extracted.

6. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to claim 2, characterized in that, Total cholesterol, triglycerides, and low-density lipoprotein cholesterol were measured weekly in the serum of transgenic mice fed the Western diet for at least 10 weeks.

7. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to claim 5, characterized in that, After extracting the mouse aorta, the aorta was frozen sectioned, stained with hematoxylin and eosin (HE) and Oil Red O, and the size of the aortic plaque was detected.

8. The method for constructing a disease model of rheumatoid arthritis combined with atherosclerosis according to any one of claims 5, characterized in that, After extracting ankle joint tissue, the mouse ankle joint was subjected to Micro-CT examination, followed by decalcification and paraffin embedding, and sectioning for HE and Fast Green staining.

9. The application of the rheumatoid arthritis combined with atherosclerosis disease model constructed by the construction method according to any one of claims 1-8 in the study of the disease mechanism of rheumatoid arthritis and / or atherosclerosis.

10. The application of the rheumatoid arthritis combined with atherosclerosis disease model constructed by the construction method according to any one of claims 1-8 in screening drugs for the treatment of rheumatoid arthritis and / or atherosclerosis.

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