A method for constructing a spontaneous rheumatoid arthritis animal model and application thereof

Abstract

The application discloses a method for constructing a spontaneous rheumatoid arthritis animal model, and the rheumatoid arthritis phenotype of the mouse is spontaneously generated by knocking out PPARgamma in the skin keratinocytes of the joint part, including joint swelling, joint synovial thickening, inflammatory cell infiltration, anti-cyclic citrullinated polypeptide antibody increase, and rheumatoid factor increase, so that the clinical symptoms of the rheumatoid arthritis are simulated, and an important tool is provided for the related research of the rheumatoid arthritis.

Description

Technical Field

[0001] This invention belongs to the technical field of animal model construction methods for diseases, specifically relating to a method for constructing an animal model of rheumatoid arthritis and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a common, chronic, systemic autoimmune disease characterized by chronic synovitis and cartilage destruction. Patients often experience persistent joint pain, swelling, and stiffness, and in severe cases, it can lead to various complications affecting the cardiovascular, pulmonary, and skeletal systems. Skin involvement is common in RA and may even be the first symptom. Skin involvement includes rheumatoid nodules, rheumatoid vasculitis, rheumatoid papules, ulcers, and pyoderma gangrenosum. The pathogenesis of RA is complex, involving the production of autoantibodies due to genetic and epigenetic factors, as well as the presence of various autoreactive T cells in the blood and synovial structures. The global incidence of RA is high, approximately 0.5%-1.0%, seriously impacting human health and quality of life. Therefore, developing cost-effective animal models that can mimic clinical symptoms is crucial for studying the pathogenesis of RA and developing safe and effective new therapies.

[0003] Currently, animal models of rheumatoid arthritis (RA) mainly include two types: inducible and transgenic mice. Inducible mouse models include collagen-induced arthritis models, antigen-induced arthritis models, collagen antibody-induced arthritis models, and proteoglycan-induced arthritis models. Transgenic mouse models include K / BxN mouse models, TNF-α (tumor necrosis factor-α) transgenic mouse models, SKG mouse models, and IL-1 (interleukin-1) receptor antagonist knockout mouse models. However, these mice are expensive, have a later onset of disease, are more susceptible to environmental factors, exhibit high heterogeneity, and some models lack rheumatoid factors and autoantibodies similar to those found in patients. Drugs developed based on these animal models cannot completely alleviate the condition of RA patients, suggesting that other factors are involved in the pathogenesis of RA. Therefore, there is an urgent need for novel spontaneous rheumatoid arthritis mouse models based on the patient's genetic background to advance research in this field and deepen our understanding of the disease.

[0004] Elevated levels of anti-keratin antibodies (AKA) are observed in some rheumatoid arthritis (RA) patients, detectable in approximately 50% of RA cases. Furthermore, elevated AKA levels can appear in the early stages of the disease. Keratin is a type of fibrous protein with supporting and protective functions, found in high concentrations in human connective tissue, particularly the skin. Keratinocytes, immune cells, and immune molecules collectively constitute the local immune microenvironment of the skin, maintaining tissue homeostasis. Although no studies have yet proven a direct link between abnormal skin function and the pathogenesis of rheumatoid arthritis, the common presence of skin involvement in RA patients suggests that imbalances in skin tissue homeostasis may be related to the autoimmune response in RA patients.

[0005] PPARγ (peroxisome proliferator-activated receptor gamma) is a key molecule in the PPAR pathway, regulating lipid synthesis and metabolism, modulating the maturation and function of immune cells, and affecting cell proliferation in tissues and organs. Its signaling dysregulation is also associated with tumorigenesis. Currently, there are no reports of constructing spontaneous RA animal models 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 to address the shortcomings of the existing technology and to provide a new model for the study and development of RA pathogenesis, pathological characteristics and treatment methods.

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

[0008] (1) Construct keratinocyte conditional PPARγ knockout mice, wherein the keratinocyte conditional PPARγ knockout mice are C57BL / 6 mice with PPARγflox / flox- / -Krt5-CreERT2+ / -;

[0009] (2) Using a gene knockout activator to knock out the PPARγ gene in keratinocytes to mediate the occurrence of disease phenotype, wherein the gene knockout activator is 4-hydroxytamoxifen; the knockout method is to apply the gene knockout activator to the skin of the knee joints, ankle joints and feet of the two lower limbs of conditionally PPARγ knockout mice after shaving.

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

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

[0012] 4-Hydroxytamoxifen is used for topical application.

[0013] Specifically, when 4-hydroxytamoxifen was used as a gene knockout activator, the skin of the knees, ankles and feet of keratinocyte conditional PPARγ knockout mice was continuously applied with 4-hydroxytamoxifen solution once a day for 4 days. The phenotype began to appear 14 days after application.

[0014] There are two application methods. Method 1 (short-term model): 4-hydroxytamoxifen solution was applied to the shaved skin of the knee, ankle, and toes of keratinocyte-conditional PPARγ knockout mice once daily for 4 consecutive days. The phenotype appeared after day 14. Method 2 (long-term model): Similarly, 4-hydroxytamoxifen solution was applied to the shaved skin of the knee, ankle, and toes of keratinocyte-conditional PPARγ knockout mice once daily for 3 consecutive days, then stopped for 7 days, then applied for another 3 days, then stopped for 7 days, and so on. One cycle was 10 days, repeated for 4 cycles. After 42 days of 4-hydroxytamoxifen application, the disease phenotype appeared.

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

[0016] Specifically, the keratinocyte conditional PPARγ knockout mouse was constructed using the following method:

[0017] PPARγflox / flox- / - and Krt5-CreERT2+ / - mice were mated to produce F1 hybrids. The heterozygous mice with the genotype PPARγflox / flox+ / -Krt5-CreERT2+ / - were then mated and bred to obtain and retain the C57BL / 6 mice with the genotype PPARγflox / flox- / -Krt5-CreERT2+ / -.

[0018] This invention further proposes the application of spontaneous RA animal models constructed using the above-described method in the study of RA disease mechanisms.

[0019] Beneficial Effects: This invention constructs a spontaneous RA animal model by knocking out PPARγ in mouse keratinocytes. This model does not require artificial induction and conditionally exhibits RA-related systemic autoimmune inflammation and joint damage, including elevated anti-cyclic citrullinated peptide (CCP) antibodies, elevated rheumatoid factor (RF), joint swelling, synovial thickening, synovial inflammatory cell infiltration, and bone destruction, without displaying systemic lupus erythematosus-related phenotypes such as persistent proteinuria and elevated antinuclear antibodies (ANA). It simulates the clinical symptoms of RA, providing an important tool for RA-related research. Compared to other RA models, this model has a genetic background rather than being drug-induced, making it highly efficient and stable. After inducing PPARγ knockout in keratinocytes, most mice in the constructed model exhibit some degree of phenotype, with low intragroup heterogeneity and strong reproducibility. Furthermore, PPARγflox / flox- / -Krt5-CreERT2+ / - mice can be continuously obtained through breeding, making it economical and convenient. Attached Figure Description

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

[0021] Figure 1 The model construction method and model validation results are presented, among which, Figure 1 A represents the modeling method; 1B represents the genotype identification of PPARγ knockout mice; 1C represents a photograph of mice after local PPARγ knockout, showing joint swelling; 1D represents the peripheral blood ANA level after local PPARγ knockout in mice; 1E represents the peripheral blood RF and anti-CCP antibody levels after local PPARγ knockout in mice; 1F represents the urinary protein level in mice after local PPARγ knockout; 1G represents the HE staining results of the knee joint in mice after local PPARγ knockout; and 1H represents the CT scan imaging results of the knee joint and foot in mice after local PPARγ knockout. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments, but it should not be construed that the scope of the present invention is limited to the following embodiments. Various substitutions and modifications made based on common technical knowledge and methods in the art without departing from the above-described technical concept of the present invention should be included within the scope of protection of the present invention.

[0023] Example 1: Rheumatoid arthritis model induced by PPARγ knockout of local keratinocytes 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), rapid identification kit for rat tail genotype (Beyotime), proteinuria test strip (Ultra), anti-CCP antibody, RF and ANA ELISA kit (Cusaibio), 4% paraformaldehyde (Biosharp), OCT embedding agent (Sakura).

[0026] 1.2 Experimental Animals

[0027] The PPARγflox / flox- / - and Krt5-CreERT2+ / - mice used in this experiment, aged approximately 6-12 weeks and weighing 20-30g, were provided by Shanghai Southern Model Biotechnology Co., Ltd. Krt5-CreERT2 mice can directionally express the CreERT2 fusion protein in keratinocytes via an endogenous promoter / enhancer element at the krt5 locus. The CreERT2 fusion protein consists of an estrogen receptor (ER) ligand-binding region mutant (ERT) and a Cre recombinase protein. Without tamoxifen induction, the CreERT2 protein is inactive in the cytoplasm; however, after tamoxifen induction, the tamoxifen metabolite 4-hydroxytamoxifen binds to ERT, allowing CreERT2 to enter the nucleus and exert Cre recombinase activity. PPARγflox / flox- / - mice, or conditional knockout mice, have loxP sites inserted at both ends of specific exons of the PPARγ gene. These loxP sites can be cleaved by active Cre recombinase. Crossbreeding Krt5-CreERT2+ / - mice with mice containing loxP flanking sequences generates offspring mice with genotypes Krt5-CreERT2+ / -PPARγflox / flox+ / - and Krt5-CreERT2+ / -PPARγflox / flox- / -. In these mice, the PPARγ gene in Krt5-expressing cells can be knocked out by tamoxifen-induced Cre-mediated gene recombination. Our constructed Krt5-CreERT2+ / -PPARγflox / flox+ / - and Krt5-CreERT2+ / -PPARγflox / flox- / - mice can precisely and directionally knock out the PPARγ gene in keratinocytes. Furthermore, compared to conventional conditional knockout mice, gene knockout timing 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 mouse model for RA disease.

[0028] C57BL / 6 mice, approximately 6-12 weeks old and weighing 20-30g, were provided by Jiangsu Jicui Pharmaceutical Co., Ltd. F1 mice were generated through hybridization, retaining the PPARγflox / flox+ / -Krt5-CreERT2+ / - mice, and further breeding yielded PPARγflox / flox- / -Krt5-CreERT2+ / - mice. The mice used in the experiments were PPARγflox / flox- / -Krt5-CreERT2+ / - mice. Housing conditions: room temperature 18-20℃, humidity 50-60%, alternating light and dark (12h), moderate light intensity, and good ventilation and cleanliness. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Institute of Dermatology, Chinese Academy of Medical Sciences (Chinese Academy of Medical Sciences Institute of Dermatology).

[0029] 2. Experimental Methods

[0030] 2.1 PPARγ knockout in mouse keratinocytes

[0031] PPARγflox / flox- / -Krt5-CreERT2+ / - mice were grouped according to requirements: 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 vortex mixer, and the mixture was sonicated in an ultrasonic bath at 37°C for 20 minutes to prepare a 4-hydroxytamoxifen solution (5 mg / mL) for use.

[0033] In the PPARγ knockout group (model group), 50 μL of 5 mg / ml 4-hydroxytamoxifen solution was applied to the skin of each lower limb knee joint, ankle joint and claw.

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

[0035] Control group: Apply equal amounts of corn oil containing 10% DMSO to the corresponding number of days and areas.

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

[0037] Using a rapid tail genotyping kit (Beyotime), genomic DNA was extracted from mouse tails according to the instructions. Fresh mouse tails were collected: scissors and forceps were rinsed with 70% ethanol before the experiment. A 0.2-1 cm section of the mouse tail tip was cut to prepare template DNA, and the PCR reaction system was prepared.

[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 is 572bp, and the size of the wild type is 445bp;

[0050] For Krt5-Cre: the size of the variant (Mutant) is 610 bp, the size of the heterozygote (Heterozygote) is 610 bp, and the size of the wild type (Wild type) is 453 bp.

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

[0052] Mice were observed for joint swelling weekly, and photographs were taken for documentation. Clean-catch midstream urine was collected from mice periodically, and urinary protein was detected using a Unirit proteinuria test strip. Peripheral blood serum ANA, anti-CCP antibody, and RF levels were detected using an ELISA kit (Cusabio).

[0053] 2.4 HE staining

[0054] Mice in the short-term model were sacrificed on day 14, and mice in the long-term model were sacrificed on day 42. Both hind limbs were harvested. The tissues were fixed in 4% paraformaldehyde, washed off with PBS, stained with hematoxylin, and washed with water until the tissue turned blue-purple. Differentiation was then performed by adding 1% hydrochloric acid-ethanol for 2 seconds until the tissue turned red, followed by washing off the hydrochloric acid-ethanol with water until the tissue turned blue. Eosin staining was then performed, the dye was washed off with water, and the tissue was covered with glycerol. The staining was confirmed under a microscope. After confirming good staining, the tissues were dehydrated, cleared, mounted, and observed under a microscope for synovial thickening, inflammatory cell infiltration, and bone destruction in the mouse knee joint.

[0055] 2.5CT scan imaging

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

[0057] 3. Experimental Results

[0058] Figure 1 A shows the modeling method. Figure 1 Figure B shows the genotyping of PPARγ conditional knockout mice in keratinocytes; Figure 1 Photo C shows a mouse image after local keratinocyte PPARγ knockout, where joint swelling is visible. Figure 1 D shows the peripheral blood ANA level after local mouse keratinocyte PPARγ knockout; Figure 1 E shows peripheral blood RF and anti-CCP antibody after local mouse keratinocyte PPARγ knockout; Figure 1 F shows the urinary protein level in mice after local PPARγ knockout of 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 results of the knee and foot of mice after local keratinocyte PPARγ knockout.

[0059] Figure 1Method A represents the modeling method. Method 1 (short-term model): Apply 4-hydroxytamoxifen solution to the shaved skin of the knee, ankle, and toes of keratinocyte-conditional PPARγ knockout mice once daily for 4 consecutive days. Method 2 (long-term model): Similarly, apply 4-hydroxytamoxifen solution to the shaved skin of the knee, ankle, and toes of keratinocyte-conditional PPARγ knockout mice once daily for 3 consecutive days, stop application for 7 days, then apply for another 3 days, stop application for 7 days, and so on. One cycle is 10 days, repeated for 4 cycles.

[0060] Figure 1 B represents the genotyping of PPARγ conditional knockout mice in keratinocytes. The target mice were PPARγ homozygous knockout mice (genotype PPARγflox / flox- / -Krt5-CreERT2+ / -, such as mouse 58) or PPARγ heterozygous knockout mice (genotype PPARγflox / flox+ / -Krt5-CreERT2+ / -, such as mice 51 and 61).

[0061] Figure 1 Photos of mice with PPARγ knockout of keratinocytes in the joint area of ​​mice on day 14 of the short-term model and day 42 of the long-term model. Erythema and swelling are visible in the toes, while no erythema or swelling is seen in the control group mice.

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

[0063] Figure 1 In mice with E-type rheumatoid arthritis (RA), peripheral blood RF and anti-CCP antibody levels were observed on days 14 and 42 of a short-term and long-term model, respectively, following PPARγ knockout of keratinocytes at the joint sites. Compared to the control group, the PPARγ knockout group showed significantly increased peripheral blood RF and anti-CCP antibody titers on days 14 and 42. These results indicate that decreased PPARγ levels in keratinocytes lead to elevated levels of diagnostic autoantibodies for rheumatoid arthritis (RA) in mice.

[0064] Figure 1 Urinary protein levels in mice were measured at days 1, 7, 12, and 14 in the short-term model and days 1, 10, 20, 30, and 42 in the long-term model after PPARγ knockout of keratinocytes in the joint area of ​​mice. Compared with the control group, there was no increase in urinary protein in the PPARγ knockout group. These results indicate that PPARγ knockout of keratinocytes in the joint area of ​​mice does not cause kidney damage.

[0065] Figure 1 HE staining results of PPARγ knockout in the keratinocytes of the joint area of ​​G mice after knee joint. Compared with the control group, the synovium of the knee joint of mice in the PPARγ knockout group was thickened, inflammatory cell infiltration was increased, and articular cartilage was damaged and disorganized.

[0066] Figure 1 H represents the CT scan results of the knee and foot joints of mice after PPARγ knockout of keratinocytes in the joint area of ​​the short-term group. Compared with the control group, the model group showed decreased bone volume fraction, reduced trabecular bone number, decreased trabecular bone thickness, and increased erosion pores.

[0067] In summary, we have established a method for constructing an animal model of spontaneous rheumatoid arthritis induced by keratinocyte PPARγ knockout, aiming to address the current challenges of large discrepancies between rheumatoid arthritis models and patients, difficulty in obtaining such models, high costs, and instability. Furthermore, this model provides new ideas and directions for exploring the pathogenesis of rheumatoid arthritis.

[0068] In the process of exploring the construction of a mouse model of rheumatoid arthritis, the following factors were found to affect the success of model construction.

[0069] ① Area of ​​4-OHT application. In previous attempts to establish this model, 4-OHT was applied to the entire skin of the hind limbs and feet of mice. This method resulted in systemic lupus erythematosus phenotypes in the mice, including elevated urinary protein, elevated antinuclear antibodies, and elevated anti-double-stranded DNA antibodies; some mice even died. Therefore, it is necessary to strictly control the area of ​​4-OHT application, limiting the application to the knee joints, ankle joints, and feet.

[0070] ② Duration of 4-OHT application. In the attempt to construct this model, we tried applying 4-OHT to the skin of both hind limbs and feet of mice continuously for 7 days. The mice began to die around the 10th day after this modeling method.

[0071] ③ Administration of 4-OHT. In our attempts to establish this model, we tried intradermal injection of 4-OHT into the knee and foot joints of mice for 7 consecutive days. However, this method resulted in mice dying around day 10. Therefore, applying an appropriate amount of 4-OHT to the knee, ankle, and foot joints of keratinocyte-conditional PPARγ knockout mice at the correct time and in the correct amount is crucial for successful model establishment. Applying too much 4-OHT over too long, or using intradermal injection, results in widespread PPARγ knockout in keratinocytes, leading to systemic lupus erythematosus phenotypes or death, thus causing model failure.

[0072] This invention provides a method for preparing a spontaneous rheumatoid arthritis animal model by knocking out PPARγ in keratinocytes. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a spontaneous rheumatoid arthritis animal model, characterized by, It comprises the following steps: (1) Constructing a keratinocyte conditional PPARγ knockout mouse, which is a C57BL / 6 mouse with a genotype of PPARγ flox / flox+ / - Krt5-CreERT2+ / -; (2) Knocking out the PPARγ gene in keratinocytes using a gene knockout activator 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 skin after shaving the knee joints, ankle joints and feet of both lower limbs of the keratinocyte conditional PPARγ knockout mouse, wherein the use of 4-hydroxytamoxifen is external application; when 4-hydroxytamoxifen is used as a gene knockout activator, 4-hydroxytamoxifen solution is continuously applied to the skin after shaving the knee joints, ankle joints and feet of both lower limbs of the keratinocyte conditional PPARγ knockout mouse once a day for 4 days, and the phenotype appears after 14 days of application, or 4-hydroxytamoxifen solution is continuously applied to the skin after shaving the knee joints, ankle joints and feet of both lower limbs of the keratinocyte conditional PPARγ knockout mouse once a day for 3 days, then stop applying for 7 days, and then continuously apply for 3 days, then stop applying for 7 days, and so on, with 10 days as one cycle, repeating for 4 cycles, and the disease phenotype appears after 42 days of applying 4-hydroxytamoxifen; The 4-hydroxytamoxifen solution is prepared according to a ratio of 50 mg 4-hydroxytamoxifen, 1 ml DMSO and 9 ml corn oil, and the dose of 4-hydroxytamoxifen solution applied to each mouse action site is 10-80 μL.

2. The construction method of claim 1, wherein, The keratinocyte conditional PPARγ knockout mouse is constructed by the following method: PPARγ flox / flox- / - and Krt5-CreERT2+ / - mice are mated to obtain hybrid offspring, and the hybrid mice with a genotype of PPARγ flox / flox+ / - Krt5-CreERT2+ / - are mated with each other to obtain and preserve C57BL / 6 mice with a genotype of PPARγ flox / flox- / - Krt5-CreERT2+ / -.

3. The application of the spontaneous rheumatoid arthritis animal model constructed by the construction method of claim 1 or 2 in the study of the disease mechanism of rheumatoid arthritis.

4. The application of the spontaneous rheumatoid arthritis animal model constructed by the construction method of claim 1 or 2 in the screening of drugs for treating rheumatoid arthritis.

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