Construction method and application of spontaneous rheumatoid arthritis animal model

Abstract

The invention discloses a method for constructing a spontaneous rheumatoid arthritis animal model, which comprises the following steps: knocking out PPAR gamma in keratinocytes of skin at joints to enable mice to spontaneously have rheumatoid arthritis phenotypes including joint swelling, joint synovial thickening, inflammatory cell infiltration, increase of anti-cyclic citrullinated polypeptide antibodies and increase of rheumatoid factors; clinical symptoms of rheumatoid arthritis are simulated, and an important tool is provided for related research of rheumatoid arthritis.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction methods, and specifically relates to a method for constructing an animal model of rheumatoid arthritis and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic, systemic autoimmune disease characterized by chronic synovitis and cartilage destruction. Patients often suffer from persistent joint pain, swelling, and stiffness. In severe cases, it can cause cardiovascular, pulmonary, and bone complications. Skin involvement is common in RA and may even be the first symptom of the disease. Skin involvement includes rheumatoid nodules, rheumatoid vasculitis, rheumatoid papules, ulcers, and pyoderma gangrenosum. The pathogenesis of RA is complex, including the production of autoantibodies caused by genetic and epigenetic factors, as well as the presence of multiple autoreactive T cells in the blood and synovial structures. The incidence of RA is high worldwide, about 0.5%-1.0%, which seriously endangers human health and quality of life. Therefore, the development of animal models that can simulate clinical symptoms and have cost advantages is of great significance for studying the pathogenesis of RA and safe and effective new therapies.

[0003] At present, animal models of rheumatoid arthritis mainly include inducible and transgenic mice. Inducible mouse models include collagen-induced arthritis model, antigen-induced arthritis model, collagen antibody-induced arthritis model, proteoglycan-induced arthritis, etc. Transgenic mouse models include K / BxN mouse model, TNF-α (tumor necrosis factor-α) transgenic mouse model, SKG mouse model, IL-1 (interleukin-1) receptor antagonist knockout mouse model, etc. However, the price of such mice is relatively high, the onset is late, they are easily affected by environmental factors, the mouse heterogeneity is large, and some mouse models do not have rheumatoid factors and autoantibodies similar to those of patients. The drugs developed based on these animal models cannot completely alleviate the condition of RA patients, suggesting that there are other factors involved in the onset of RA. Therefore, there is an urgent need for new spontaneous rheumatoid arthritis mouse models based on the genetic background of patients to promote research in this field and deepen the understanding of the disease.

[0004] Some patients with rheumatoid arthritis have elevated anti-keratin antibodies (AKA), which can be detected in about 50% of patients with rheumatoid arthritis. And the increase in AKA can appear in the early stages of the patient. Keratin is a type of fibrous protein with supporting and protective functions, and its content is high in human connective tissue, especially the skin. Keratinocytes, immune cells and immune molecules together constitute the local immune microenvironment of the skin to maintain tissue homeostasis. Although no studies have proven a direct link between skin dysfunction and the onset of rheumatoid arthritis, skin involvement is common in patients with rheumatoid arthritis, suggesting that disorders of skin tissue homeostasis may be related to the autoimmune response of patients with rheumatoid arthritis.

[0005] PPARγ (Peroxisome proliferator-activated receptor gamma) is a key molecule in the PPAR pathway, which can regulate the synthesis and metabolism of lipids, the maturation and function of immune cells, and affect the cell proliferation of tissues and organs. Its signal dysregulation is also related to tumor formation. So far, there has been no report on the construction of a spontaneous RA animal model using keratinocyte conditional PPARγ knockout mice. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a new spontaneous RA animal model for the study and development of the pathogenesis, pathological characteristics and treatment methods of RA in view of the deficiencies of the prior art.

[0007] To achieve the above technical objectives, the present invention discloses a method for constructing a spontaneous RA animal model, comprising the following steps:

[0008] (1) constructing a keratinocyte conditional PPARγ knockout mouse, wherein the keratinocyte conditional PPARγ knockout mouse is a PPARγflox / flox- / -Krt5-CreERT2+ / - C57BL / 6 mouse;

[0009] (2) Using a gene knockout activator to knock out the PPARγ gene in keratinocytes to mediate the occurrence of disease phenotypes, wherein the gene knockout activator is 4-hydroxytamoxifen; the knockout method is to apply the gene knockout activator to the shaved skin of the knee joints, ankle joints and feet of the keratinocyte conditional PPARγ knockout mice.

[0010] The gene knockout activator is a compound that can bind to the estrogen receptor mutant ERT after metabolism, so that CreERT2 can exert Cre recombinase activity to perform gene knockout.

[0011] In one embodiment, the gene knockout activator is tamoxifen or 4-hydroxytamoxifen.

[0012] Among them, 4-hydroxytamoxifen is used for external application.

[0013] Specifically, when 4-hydroxytamoxifen was used as a gene knockout activator, 4-hydroxytamoxifen solution was applied to the shaved skin of both knee joints, both ankle joints and both feet of keratinocyte conditional PPARγ knockout mice for 4 consecutive days, once a day, and the phenotype began to appear after 14 days after application.

[0014] There are two application methods. Method 1 (short-term model): Apply 4-hydroxytamoxifen solution to the shaved skin of the knees, ankles and toes of keratinocyte conditional PPARγ knockout mice for 4 consecutive days, once a day. The phenotype begins to appear after 14 days of application; Method 2 (long-term model): Similarly, apply 4-hydroxytamoxifen solution to the shaved skin of the knees, ankles and toes of keratinocyte conditional PPARγ knockout mice for 3 consecutive days, once a day, stop applying for 7 days, then apply for 3 consecutive days and then stop applying for 7 days, and so on. 10 days is one cycle, repeated 4 cycles, repeated 4 cycles, and disease phenotypes appear 42 days after applying 4-hydroxytamoxifen.

[0015] The 4-hydroxytamoxifen solution is prepared according to the ratio of 50 mg 4-hydroxytamoxifen, 1 ml DMSO, and 9 ml corn oil; the dosage of the 4-hydroxytamoxifen solution applied externally to the mice each time is 10-80 μL.

[0016] Specifically, the keratinocyte conditional PPARγ knockout mouse is constructed by the following method:

[0017] PPARγflox / flox- / - and Krt5-CreERT2+ / - mice were mated to obtain the first generation of hybrids, and heterozygous mice with the genotype of PPARγflox / flox+ / -Krt5-CreERT2+ / - were mated with each other to obtain and retain C57BL / 6 mice with the genotype of PPARγflox / flox- / -Krt5-CreERT2+ / -.

[0018] The present invention further proposes the application of the spontaneous RA animal model constructed by the above construction method in studying the disease mechanism of RA.

[0019] Beneficial effects: The present invention constructs a spontaneous RA animal model by knocking out PPARγ in mouse keratinocytes. It does not require artificial induction and can conditionally show RA-related systemic autoimmune inflammation and joint damage, including increased anti-cyclic citrullinated peptide (CCP) antibodies, increased rheumatoid factor (RF), joint swelling, synovial thickening, synovial inflammatory cell infiltration, and bone destruction, without the appearance of systemic lupus erythematosus-related phenotypes, such as persistent urine protein and increased antinuclear antibodies (ANA). It simulates the clinical symptoms of RA and provides an important tool for related research on RA. Compared with other RA models, this model has a genetic background rather than drug induction, and is efficient and stable. After inducing the knockout of PPARγ in keratinocytes, most of the mice constructed in the model will show a certain degree of phenotype, with low intra-group heterogeneity and strong repeatability. In addition, PPARγflox / flox- / -Krt5-CreERT2+ / - mice can be continuously obtained through breeding, which is economical and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 is the model construction method and model verification results, among which, Figure 1 A is the membrane-making method, 1B is the genotype identification of keratinocyte PPARγ conditional knockout mice; 1C is a photo of mice after local keratinocyte PPARγ knockout, and joint swelling can be seen; 1D is the peripheral blood ANA level after local mouse keratinocyte PPARγ knockout; 1E is the peripheral blood RF and anti-CCP antibody content after local mouse keratinocyte PPARγ knockout; 1F is the urine protein level of mice after local mouse keratinocyte PPARγ knockout; 1G is the HE staining result of the knee joint of mice after local keratinocyte PPARγ knockout; 1H is the CT scan imaging result of the knee joint and foot of mice after local keratinocyte PPARγ knockout. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the embodiments, but it should not be understood that the above subject scope of the present invention is limited to the following embodiments. Without departing from the above technical ideas of the present invention, various substitutions and changes are made according to the common technical knowledge and customary methods in the field, which should all be included in the protection scope of the present invention.

[0023] Example 1 Rheumatoid arthritis model induced by local keratinocyte PPARγ knockout activated by 4-hydroxytamoxifen application.

[0024] 1. Experimental Materials

[0025] 1. Experimental drugs and reagents: DMSO (Sigma-Aldrich), 4-hydroxytamoxifen (Sigma-Aldrich), corn oil (Beyotime), mouse tail genotype rapid identification kit (Beyotime), proteinuria test strips (Ulite), anti-CCP antibody, RF and ANA ELISA kits (Cusaibio), 4% paraformaldehyde (Biosharp), OCT embedding medium (Sakura).

[0026] 1.2 Experimental animals

[0027] The PPARγflox / flox- / - and Krt5-CreERT2+ / - mice used in this experiment were about 6-12 weeks old and weighed 20-30g, and were provided by Shanghai Model Organisms Technology Co., Ltd. Krt5-CreERT2 mice can express CreERT2 fusion protein in keratinocytes through the endogenous promoter / enhancer element of the krt5 locus. The CreERT2 fusion protein is composed of the ligand binding region mutant (ERT) of the estrogen receptor (ER) and the Cre recombinase protein. In the absence of tamoxifen induction, the CreERT2 protein is inactive in the cytoplasm; after tamoxifen induction, the metabolite of tamoxifen, 4-hydroxytamoxifen, binds to ERT, allowing CreERT2 to enter the nucleus and exert the recombinase activity of Cre. PPARγflox / flox- / - mice are PPARγ conditional knockout mice, in which loxP sites are inserted at both ends of specific exons of the PPARγ gene, and the loxP sites can be cut by active Cre recombinase. Krt5-CreERT2+ / - mice are mated with mice containing loxP flanking sequences to generate offspring mice with the genotype of Krt5-CreERT2+ / -PPARγflox / flox+ / - and Krt5-CreERT2+ / -PPARγflox / flox- / -. The PPARγ gene in cells expressing Krt5 in these mice can be knocked out by tamoxifen-induced Cre-mediated gene recombination. The Krt5-CreERT2+ / -PPARγflox / flox+ / - and Krt5-CreERT2+ / -PPARγflox / flox- / - mice we constructed can precisely knock out the PPARγ gene in keratinocytes. Compared with general conditional knockout mice, the timing of gene knockout can be precisely controlled, and the phenotypes of mice before and after gene knockout can be compared on the same individual, which can simulate the disease process to the greatest extent, making it an ideal RA disease model mouse.

[0028] C57BL / 6 mice, about 6-12 weeks old and weighing 20-30g, were provided by Jiangsu Jicui Pharmaceutical Co., Ltd. The first generation of hybridization was generated, and PPARγflox / flox+ / -Krt5-CreERT2+ / - mice were retained, and PPARγflox / flox- / -Krt5-CreERT2+ / - mice were obtained by further breeding. The mice used in the experiment were PPARγflox / flox- / -Krt5-CreERT2+ / - mice. Housing conditions: room temperature 18-20℃, humidity 50-60%, alternating light and dark (12h), moderate lightness, and good ventilation and cleanliness. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Dermatology Hospital of the Chinese Academy of Medical Sciences (Institute of Dermatology, Chinese Academy of Medical Sciences).

[0029] 2. Experimental Methods

[0030] 2.1 PPARγ knockout in mouse keratinocytes

[0031] PPARγflox / flox- / -Krt5-CreERT2+ / - mice were divided into groups according to the needs: control group, PPARγ

[0032] Knockout group (model group: long-term model, short-term model). 50 mg of 4-hydroxytamoxifen was mixed in 1 ml of DMSO, 9 ml of corn oil was added, the suspension was thoroughly mixed using a vortexer, and sonicated in an ultrasonic bath at 37° C. for 20 minutes to prepare a 4-hydroxytamoxifen solution (5 mg / mL) to be used.

[0033] In the PPARγ knockout group (model group), 50ul of 5mg / ml 4-hydroxytamoxifen solution was applied to the skin of each part of the knee joints, ankle joints and paws of both lower limbs.

[0034] Model group: ① Short-term model: Apply the corresponding solution to the corresponding part for 4 consecutive days, once a day. ② Long-term model: Apply 4-hydroxytamoxifen solution for 3 consecutive days, once a day, stop applying for 7 days, then apply for 3 consecutive days, then stop applying for 7 days, and so on. 10 days is one cycle, and repeat 4 cycles.

[0035] Control group: Apply an equal amount of corn oil containing 10% DMSO on the corresponding days and parts.

[0036] 2.2 PCR identification of keratinocyte PPARγ conditional knockout mouse genotype

[0037] Use the Mouse Tail Genotype Rapid Identification Kit (Beyotime) according to the instructions to extract mouse tail genomic DNA. Take a fresh mouse tail: Rinse the scissors and tweezers with 70% ethanol before the experiment. Cut 0.2-1cm of the mouse tail tip to prepare template DNA and prepare the PCR reaction system:

[0038] Table 1 PCR reaction system

[0039]

[0040]

[0041] Table 2 PCR reaction parameters

[0042]

[0043] After the PCR reaction, agarose gel electrophoresis was performed to detect the mouse genotype. The primer sequences are as follows:

[0044] PPARγflox / flox primer 1: 5′-CTTCCCCTTCCCCAAAATGAGTC-3′;

[0045] PPARγflox / flox primer 2: 5′-TCTGTGGCTGGACTACAGGA-3′;

[0046] Krt5-e (2A-CreERT2) Primer 1: 5′-GTGGCTTACATTCTGCAACATTTT-3′;

[0047] Krt5-e (2A-CreERT2) Primer 2: 5′-GGCCCACGCTTCACCAG-3′;

[0048] Krt5-e (2A-CreERT2) Primer 3: 5′-GGATCCGCCGCATAACCAGT-3′.

[0049] Among them, for PPARγflox: the size of the variant (Mutant) is 572bp, the size of the heterozygote (Heterozygote) is 572bp, and the wild type (Wild type) is 445bp;

[0050] For Krt5-Cre: the size of the mutant is 610 bp, the size of the heterozygote is 610 bp, and the size of the wild type is 453 bp.

[0051] 2.3 Monitoring of joint swelling, urine protein and autoantibodies in mice

[0052] The joint swelling of mice was observed weekly and photographed. Clean midstream urine of mice was collected regularly and urine protein was tested using the Ulite protein urine test paper. ELISA kits (Cusabio) were used to detect the levels of ANA, anti-CCP antibodies, and RF in peripheral blood serum.

[0053] 2.4 HE staining

[0054] Mice were killed on the 14th day for the short-term model and on the 42nd day for the long-term model. The lower limbs of the mice were removed. The mice were fixed with 4% paraformaldehyde, the fixative was washed off with PBS, and hematoxylin was added for staining. The tissue was washed with water until it turned blue-purple. 1% hydrochloric acid ethanol was added for differentiation for 2 seconds. When the tissue turned red, the hydrochloric acid ethanol was washed off with water until the tissue turned blue. Eosin staining was performed, the dye was washed off with water, and the tissue was covered with glycerol. The staining was confirmed under a microscope. After confirming that the staining was intact, dehydrated, transparent, and sealed, the thickening of the synovium, infiltration of inflammatory cells, and bone destruction of the mouse knee joint were observed under a microscope.

[0055] 2.5 CT Scanning

[0056] The mice were killed on the 14th day in the short-term model and on the 42nd day in the long-term model, and the lower limbs of the mice were removed. Micro-CT was used to perform structural imaging of the lower limbs of the mice to observe bone destruction and bone loss.

[0057] 3. Experimental results

[0058] Figure 1 A shows the modeling method. Figure 1 Panel B shows the genotype identification of keratinocyte PPARγ conditional knockout mice; Figure 1 C shows a photo of mice after local keratinocyte PPARγ knockout, showing joint swelling; Figure 1 D shows the peripheral blood ANA level after local knockout of PPARγ in mouse keratinocytes; Figure 1 E shows peripheral blood RF and anti-CCP antibody after local knockout of PPARγ in mouse keratinocytes; Figure 1 F shows the urine protein level of mice after local knockout of PPARγ in mouse keratinocytes; Figure 1 G shows the HE staining results of the mouse knee joint after local keratinocyte PPARγ knockout; Figure 1 H shows the CT scan imaging results of the knee joint and foot of mice after local keratinocyte PPARγ knockout.

[0059] Figure 1A is the modeling method. Method 1 (short-term model): 4-hydroxytamoxifen solution was applied to the shaved skin of the knees, ankles and toes of keratinocyte conditional PPARγ knockout mice for 4 consecutive days, once a day; Method 2 (long-term model): Similarly, 4-hydroxytamoxifen solution was applied to the shaved skin of the knees, ankles and toes of keratinocyte conditional PPARγ knockout mice for 3 consecutive days, once a day, and then stopped for 7 days, then continued to apply for 3 days and then stopped for 7 days, and so on. 10 days is one cycle, repeated 4 cycles, and repeated 4 cycles.

[0060] Figure 1 B is the genotype identification of keratinocyte PPARγ conditional knockout mice. The target mice are PPARγ homozygous knockout mice (genotype is PPARγflox / flox- / -Krt5-CreERT2+ / -, such as No. 58) or PPARγ heterozygous knockout mice (genotype is PPARγflox / flox+ / -Krt5-CreERT2+ / -, such as No. 51 and No. 61).

[0061] Figure 1 C is a photo of mice on the 14th day of the short-term model and the 42nd day of the long-term model of PPARγ knockout in the skin keratinocytes of the mouse joints. Erythema and swelling can be seen in the toes, while there is no erythema or swelling in the control group mice.

[0062] Figure 1 D. Peripheral blood antinuclear antibody levels after PPARγ knockout in mouse joint skin keratinocytes on day 14 of the short-term model and day 42 of the long-term model. Compared with the control group, peripheral blood antinuclear antibodies in the PPARγ knockout group mice did not increase on days 14 and 42 of modeling, indicating that this model does not meet the diagnostic criteria for systemic lupus erythematosus arthritis.

[0063] Figure 1 E. Peripheral blood RF and anti-CCP antibodies after PPARγ knockout in keratinocytes of the joints of mice on day 14 of the short-term model and day 42 of the long-term model. Compared with the control group, the peripheral blood RF and anti-CCP antibody titers of the PPARγ knockout group mice increased significantly on day 14 and day 42 of modeling. The above results indicate that the reduction of PPARγ in keratinocytes will cause the mice to have increased diagnostic autoantibodies for RA.

[0064] Figure 1 F Mouse joint skin keratinocyte PPARγ knockout mice urinary protein levels at 1, 7, 12, 14 days in the short-term model and 1, 10, 20, 30, 42 days in the long-term model. Compared with the control group, the urinary protein of mice in the PPARγ knockout group did not increase. The above results indicate that the knockout of PPARγ in the skin keratinocytes of the mouse joints will not cause kidney damage in mice.

[0065] Figure 1 G HE staining results of knee joints after PPARγ knockout in skin keratinocytes of mice joints. Compared with the control group, the synovium of the knee joints of mice in the PPARγ knockout group was thickened, the infiltration of inflammatory cells increased, and the articular cartilage was damaged and disordered.

[0066] Figure 1 H is the CT scan imaging result of knee joint and foot of mice after PPARγ knockout in skin keratinocytes of joints of mice in short-term group. Compared with the control group, the bone volume fraction, number of trabeculae, thickness of trabeculae and number of corrosion holes in the modeling group were reduced.

[0067] In summary, we have established a method for constructing a spontaneous rheumatoid arthritis animal model induced by keratinocyte PPARγ knockout, in order to solve the difficulties of the current rheumatoid arthritis model, such as the large gap between the current model and patients, the difficulty in obtaining, the high price, and the instability. This model also provides new ideas and directions for exploring the pathogenesis of rheumatoid arthritis.

[0068] In the process of exploring the construction of rheumatoid arthritis mouse model, it was found that the following factors would also affect the success of model construction.

[0069] ① 4-OHT application area. In the attempt to construct this model, 4-OHT was applied to the entire skin of the lower limbs and feet of mice. This modeling method will cause the mice to develop the phenotype of systemic lupus erythematosus, including increased urine protein, increased antinuclear antibodies, and increased anti-double-stranded DNA antibodies, and some mice will die. Therefore, it is necessary to strictly control the area of ​​4-OHT application and limit the application location to the knee joints, ankle joints, and feet.

[0070] ② Time of applying 4-OHT. In the attempt to construct this model, an attempt was made to apply 4-OHT to the entire skin of both lower limbs and feet of mice for 7 consecutive days. In this modeling method, mice began to die around the 10th day.

[0071] ③The administration method of 4-OHT. In the attempt to construct this model, 4-OHT was injected intradermally into the knee joints and foot skin of mice for 7 consecutive days. The mice began to die around the 10th day in this modeling method. Therefore, applying an appropriate amount of 4-OHT only on the knee joints, ankle joints and feet of keratinocyte conditional PPARγ knockout mice and at the right time is the key factor for successful modeling. When the application area is too large, the time is too long, or intradermal injection is used, the knockout area of ​​mouse PPARγ in keratinocytes is relatively wide, which will cause the mice to develop the phenotype of systemic lupus erythematosus or die, resulting in modeling failure.

[0072] The present invention provides a method and idea for preparing a spontaneous rheumatoid arthritis animal model by knocking out keratinocyte PPARγ. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for constructing a spontaneous rheumatoid arthritis animal model, characterized in that: The steps include: (1) constructing a keratinocyte conditional PPARγ knockout mouse, wherein the keratinocyte conditional PPARγ knockout mouse is a C57BL / 6 mouse with a genotype of PPARγ flox / flox+ / - Krt5-CreERT2+ / -; (2) Using a gene knockout activator to knock out the PPARγ gene in keratinocytes to mediate the occurrence of disease phenotypes, wherein the gene knockout activator is 4-hydroxytamoxifen; the knockout method is to apply the gene knockout activator to the shaved skin of both knee joints, both ankle joints and both feet of keratinocyte conditional PPARγ knockout mice.

2. The construction method according to claim 1, characterized in that: 4-Hydroxytamoxifen is applied topically.

3. The construction method according to claim 1, characterized in that: When 4-hydroxytamoxifen was used as a gene knockout activator, 4-hydroxytamoxifen solution was applied to the shaved skin of both knee joints, both ankle joints and both feet of keratinocyte conditional PPARγ knockout mice for 4 consecutive days, once a day, and the phenotype began to appear after 14 days after application.

4. The construction method according to claim 1, characterized in that: 4-Hydroxytamoxifen solution was applied to the shaved skin of the knee joints, ankle joints and feet of both lower limbs of keratinocyte conditional PPARγ knockout mice once a day for 3 consecutive days, and then stopped for 7 days. Then, it was applied for 3 consecutive days and then stopped for 7 days, and so on. The cycle was repeated for 4 times with 10 days as one cycle. The disease phenotype appeared 42 days after the application of 4-hydroxytamoxifen.

5. The construction method according to claim 3 or 4, characterized in that: The 4-hydroxytamoxifen solution is prepared according to the ratio of 50 mg 4-hydroxytamoxifen, 1 ml DMSO, and 9 ml corn oil. The dosage of the 4-hydroxytamoxifen solution evenly applied to a single action site of the mouse each time is 10-80 μL.

6. The construction method according to claim 1, characterized in that: The keratinocyte conditional PPARγ knockout mouse was constructed by the following method: PPARγ flox / flox- / - and Krt5-CreERT2+ / - mice were mated to obtain the first generation of hybrids, and heterozygous mice with the genotype of PPARγ flox / flox+ / - Krt5-CreERT2+ / - were mated with each other to obtain and retain C57BL / 6 mice with the genotype of PPARγ flox / flox- / - Krt5-CreERT2+ / -.

7. Application of the spontaneous rheumatoid arthritis animal model constructed by the construction method according to any one of claims 1 to 6 in studying the disease mechanism of rheumatoid arthritis.

8. Use of the spontaneous rheumatoid arthritis animal model constructed by the construction method according to any one of claims 1 to 6 in screening drugs for treating rheumatoid arthritis.

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