A method for constructing an animal model of rheumatoid arthritis

Abstract

The application belongs to the technical field of disease animal model construction method, and particularly relates to a construction of an animal model of rheumatoid arthritis. The application uses SIRT3 knockout female mice, induces an arthritis model through mBSA antigen, and after primary immunization and secondary immunization, the rheumatoid arthritis phenotype appears one week after the secondary immunization. The mouse obtained in the application can appear knee joint injury, local joint inflammatory cell infiltration and obvious increase of inflammatory factors after one to two weeks of modeling. The rheumatoid arthritis mouse model obtained in the application has short modeling cycle, high and stable incidence rate, and clear pathogenesis, and is convenient for researchers to widely use.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of disease animal model construction method, and particularly relates to construction of an animal model of rheumatoid arthritis. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a common, chronic, and autoimmune disease mainly caused by inflammatory synovitis. The disease is recurrent and persistent, and is one of the main causes of human labor loss and disability. With the progress of research on the pathogenesis of RA, many new therapeutic drugs have been developed and applied. The development and application of these drugs have improved the condition of patients and the prognosis of the disease to some extent, but still cannot fundamentally prevent the inflammatory joint destruction of RA.

[0003] At present, researchers generally believe that the abnormal activation of T / B and other immune cells and the lack of immune tolerance lead to an increase in self-reactive cells, which is one of the important pathogenesis of RA. More and more studies have proved that the abnormal metabolic state of cells plays an important role in the occurrence of RA in the process of T / B cell activation. After stimulation, the metabolic state of lymphocytes changes from the metabolic pattern of aerobic metabolism (resting lymphocytes) to glycolysis metabolism (activated state). Studies have shown that the metabolic state of T cells in RA patients is significantly abnormal. Compared with normal people, the glucose utilization, lactic acid production, and intracellular ATP generation of T cells in RA patients are significantly reduced after stimulation, and the apoptosis is increased. However, the regulatory mechanism of these metabolic abnormalities is still unclear.

[0004] Current rheumatoid arthritis model, generally using wild type mouse collagen induced animal model, or wild type mouse antigen induced model, these models of rheumatoid arthritis phenotype difference is bigger, especially for C57BL / 6 background mice, rheumatoid arthritis model has natural resistance, the degree of morbidity is weak after modeling, and the incidence rate reported in different literatures is not consistent, which causes great trouble to researchers. Yajaira B. Guedez et al. found that the incidence rate of collagen-induced arthritis model of wild type C57BL / 6 background mice was only 14%, and the onset time was long, and the phenotype appeared 51 days after immunization. C-Q Chu et al. reported that the incidence rate of collagen-induced arthritis model of wild type C57BL / 6 background mice was about 10%, and the onset time was 65 days after immunization. Ian K. Campbell1 et al. reported that the incidence rate of collagen-induced arthritis model of wild type C57BL / 6 background mice was 63%-70%, but the onset time was 51-60 days after immunization. In addition, K. Oleinika et al. showed that the incidence rate of antigen-induced arthritis model of wild type C57BL / 6 background mice was 44%, and the phenotype appeared within 1 week after secondary immunization. Even so, the incidence rate is low.

[0005] Therefore, the incidence rate of rheumatoid arthritis mouse model, especially C57BL / 6 background mice, is low after modeling, the degree of morbidity is weak, and the experimental process is not easy to control, which brings difficulties to the study of the pathogenesis of the disease. SUMMARY

[0006] In order to solve the above problems, the present application provides a new rheumatoid arthritis model: the cause is clear, mainly causes CD4+ T lymphocyte abnormality, and the experimental period is short, which provides a new animal model for the study of the pathogenesis of rheumatoid arthritis.

[0007] The present application provides a construction method of a new animal model of rheumatoid arthritis, which uses C57BL / 6 background SIRT3 knockout mice to induce rheumatoid arthritis modeling by mBSA antigen.

[0008] In one embodiment of the present application, the mBSA antigen-induced rheumatoid arthritis model includes:

[0009] 1) Primary immunization: on day 1, methylated bovine serum albumin (mBSA) is dissolved in PBS or physiological saline, filtered after dissolution, and configured into a solution of 2 μg / μl. The concentration of complete Freund's adjuvant (CFA) is 5 mg / ml, the mixed mBSA and CFA is 1:1, the final concentration of mBSA is 1 μg / μl, and the mixed solution is injected into the root of the mouse tail, 200 μl per mouse. The total dose of mBSA injected into each mouse is 200 μg.

[0010] 2) Second immunization: On day 7, mice are injected with mBSA in the knee joint, mBSA is dissolved in PBS or normal saline, filtered after dissolution, configured into a solution of 20 μg / μl, and injected with 10 μl. The needle is inserted from the side of the knee joint and injected into the knee joint cavity. After the second immunization, the movement of the mice can be observed, and the observation is performed for 7 days; on day 14, the modeling is completed, the knee joint destruction of the mice is observed by imaging, the knee joint is taken for fixation and sectioning, and staining is performed, so that the knee joint damage and inflammatory infiltration of the mice can be observed.

[0011] In the formula, the SIRT3 knockout mouse for inducing rheumatoid arthritis is a female mouse after 8 weeks of adulthood.

[0012] In the formula, when the background of the SIRT3 knockout mouse is not a C57BL / 6 background, the SIRT3 knockout mouse is continuously backcrossed with a C57BL / 6 strain mouse for more than 10 generations, so that the SIRT3 knockout mouse becomes a C57BL / 6 background.

[0013] In the formula, the SIRT3 knockout mouse is constructed by knocking out exon 2-3 of the mouse SIRT3 gene. The NCBI ID of the mouse SIRT3 gene is 64384.

[0014] In the formula, the specific construction method of the SIRT3 knockout mouse is shown in the website of Jacson lab, http: / / jaxmice.jax.org / strain / 012755.html.

[0015] The mouse used in the application is a SIRT3 gene knockout mouse with exon 2-3. SIRT3 is a member of the NAD+-dependent class III histone deacetylase Sirtuins family, and plays an important role in regulating fatty acid metabolism, sugar metabolism and other metabolic processes. Our research shows that SIRT3 knockout can cause abnormal glycolysis metabolism of CD4+ T cells, insufficient intracellular ATP generation, increase of T cell apoptosis, and thus promote the occurrence and development of rheumatoid arthritis.

[0016] The rheumatoid arthritis mouse model obtained by the application has a short modeling cycle, high and stable incidence rate, and clear pathogenesis, and is convenient for researchers to widely use. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The SIRT3 knockout mouse gene is identified. It can be seen that SIRT3 has basically no expression in the knockout mouse.

[0018] Figure 2SIRT3 knockout mice had more severe knee joint swelling and paresis than wild type mice after the same method of inducing arthritis model.

[0019] Figure 3 SIRT3 knockout mice had a significantly increased incidence of rheumatoid arthritis than wild type mice after the same method of inducing arthritis model.

[0020] Figure 4 SIRT3 knockout mice had more obvious inflammatory cell infiltration in the knee joint than wild type mice after the same method of inducing arthritis model, as shown by HE staining.

[0021] Figure 5 SIRT3 knockout mice had more obvious inflammatory cell infiltration in the knee joint than wild type mice after the same method of inducing arthritis model, as shown by statistical chart. Female mice = 6-8. The data of all scatter plots is shown by mean ± SEM. * P<0.05; ** P<0.01; *** P<0.001.

[0022] Figure 6 SIRT3 knockout mice had more obvious cartilage destruction in the knee joint than wild type mice after the same method of inducing arthritis model, as shown by Papanicolaou green staining results.

[0023] Figure 7 SIRT3 knockout mice had more obvious cartilage destruction in the knee joint than wild type mice after the same method of inducing arthritis model, as shown by statistical chart. Female mice = 6-8. The data of all scatter plots is shown by mean ± SEM. * P<0.05; ** P<0.01; *** P<0.001.

[0024] Figure 8 SIRT3 knockout mice had more obvious cartilage destruction in the knee joint than wild type mice after the same method of inducing arthritis model, as shown by CT scan results.

[0025] Figure 9 SIRT3 knockout mice had more obvious decrease in bone density than wild type mice after the same method of inducing arthritis model, as shown by CT scan results.

[0026] Figure 10For the statistical chart, the bone density of the SIRT3 knockout mouse arthritis induction model decreased more significantly than that of the wild type mouse induced by the same method. Female mice = 6-8. The data of all scatter plots are shown by mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

[0027] Figure 11 For the result of fast green staining, the rheumatoid arthritis phenotype (knee joint destruction) caused by SIRT3 deletion disappeared after CD4+T cells were removed from the mice.

[0028] Figure 12 For the HE staining showing the inflammatory cell infiltration of the knee joint of the mice, the rheumatoid arthritis phenotype (inflammatory cell infiltration) caused by SIRT3 deletion disappeared after CD4+T cells were removed from the mice. Figure 10 , 11 shows that SIRT3 mainly acts through mouse CD4+T cells.

[0029] Figure 13 For the flow cytometry detection of CD4+T cell removal, the CD4+T cells in the mice treated with CD4 removal antibody decreased or even disappeared.

[0030] Figure 14 For the statistical chart, the inflammatory factor of the SIRT3 knockout mouse arthritis induction model increased more significantly than that of the wild type mouse induced by the same method. Female mice = 5-10. The data of all scatter plots are shown by mean ± SEM. *P<0.05; **P<0.01; ***P<0.001.

[0031] Figure 15 For knocking down SIRT3 in human CD4+T cells, T cell apoptosis increased.

[0032] Figure 16 For the statistical chart, knocking down SIRT3 in human CD4+T cells increased T cell apoptosis. n=5. The data of all scatter plots are shown by mean ± SEM. *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application.

[0034] Example 1 SIRT3 knockout mouse gene identification scheme

[0035] The SIRT3 knockout mouse is a whole-body knockout mouse (purchased from Jacson lab, item number: 012755, background: 129), and the identification method is to cut the mouse claws for identification. The identification result is as followsFigure 1 .

[0036] Gene information: SIRT3 gene is located in mouse chromosome 11.

[0037] Identification method: cut mouse toes in tissue digestion solution (29.5 μL tissue digestion solution + 0.5 μL proteinase K), high-speed centrifugation to fully infiltrate the tissue in the digestion solution, digestion at 55℃ overnight, boiling for 10 min to inactivate DNA enzyme, centrifugation at 4℃-12,000 rpm for 10 min, and the supernatant can be used as a PCR template. The PCR product is subjected to agarose gel electrophoresis.

[0038] Tissue digestion solution composition: KCl 500 mM, Tris-HCl 100 mM, gelatin (Sigma G2500) 0.1 mg / mL, NP-40 0.45%, Tween-20 0.45%, proteinase K (added before use) 0.5 mg / mL.

[0039] Identification primer:

[0040] F1, 5'-CTT CTG CGG CTC TAT ACA CAG-3' (common primer);

[0041] R1, 5'-TGC AAC AAG GCT TTA TCT TCC-3' (wild type);

[0042] R2, 5'-TAC TGA ATA TCA GTG GGA ACG-3' (after knockout).

[0043] The identification results of SIRT3 knockout mice are shown in Table 1. Figure 1 SIRT3 is almost not expressed in knockout mice.

[0044] Example 2: Phenotype identification of SIRT3 knockout mice with rheumatoid arthritis

[0045] The SIRT3 knockout mice of the present application are purchased from Jacson lab (item number: 012755), and the 2-3thexons of the SIRT3 gene of the knockout mice are knocked out to construct the SIRT3 knockout mice. The mice are of 129 background, and after being received, they are continuously backcrossed with C57BL / 6 strain mice for more than 10 generations, so that the SIRT3 knockout mice become C57BL / 6 background.

[0046] After the SIRT3 knockout mice are 8 weeks old, the antigen-induced rheumatoid arthritis model is carried out. The SIRT3 knockout female mice of the present application are selected for model induction, and the specific construction method of the antigen-induced rheumatoid arthritis model is as follows:

[0047] 1) First immunization, day 1, methylated bovine serum albumin (mBSA) was dissolved in PBS or normal saline, after dissolution, filtered, configured into a solution of 2 μg / μl, the concentration of complete Freund's adjuvant (CFA) was 5 mg / ml, the prepared mBSA was mixed with CFA at a ratio of 1:1, the final concentration of mBSA was 1 μg / μl, and the mixture was injected into the root of the mouse tail, 200 μl of the mixture was injected into the root of each mouse tail. The total dose of mBSA injected into each mouse was 200 μg.

[0048] 2) Second immunization, day 7, mBSA was injected into the knee joint of the mouse, mBSA was dissolved in PBS or normal saline, after dissolution, filtered, configured into a solution of 20 μg / μl, and 10 μl was injected. The needle was inserted from the side of the knee joint and injected into the knee joint space. After the second immunization, the movement of the mouse was observed, and the observation was continued for 7 days.

[0049] On day 14, the modeling was completed, the knee joint destruction of the mouse was observed by imaging, the knee joint was taken for fixation, sectioning and staining, and the knee joint damage and inflammatory infiltration of the mouse were observed. The arthritic phenotype was observed obviously 7-14 days after modeling. Figures 2-15 ).

[0050] Joint swelling and limp score: female SIRT3-KO mice or WT C57 / BL6 mice (8-12 weeks old) were divided into AIA group and control group. The AIA group was immunized with 200 μl CFA mixed with 200 μg mBSA subcutaneously at the bottom of the tail, and was immunized again 7 days later. The mice received 10 μl mBSA (20 μg / μl) intra-articular injection in the right knee and PBS intra-articular injection in the left knee as a control. The control group was injected with PBS in both legs. The joint size was measured using a caliper (POCO 2T; Kroeplin), and the percentage increase in swelling of the mBSA-injected knee compared with the control knee was calculated for 7 consecutive days after the onset of the disease (after the second immunization). The limp score criteria are as follows: 4 points, unable to walk; 3 points, walking on three legs; 2 points, walking on a knee joint inflamed leg; 1 point, walking on a knee joint inflamed leg after pressure on the knee joint; and 0 indicates normal walking.

[0051] Female SIRT3-KO or WT mice were divided into AIA group (20 in each group) and control group (10 in each group), and it was found that the knee joint swelling and limp of the SIRT3 knockout mouse arthritic induction model were more severe than those of the wild type mouse induced model by the same method Figure 2 ), and 16 of the 20 SIRT3 knockout mice developed rheumatoid arthritis phenotype after AIA modeling, with a morbidity rate of 80%, while only 9 of the 20 WT mice developed rheumatoid arthritis phenotype after modeling (morbidity rate 45%), indicating that the morbidity rate of SIRT3 knockout mouse AIA modeling was significantly higher than that of wild type mouse modeling Figure 3).

[0052] Staining and Safranin O-fast green staining: At day 14 of modeling, the knee joints were harvested from the mice in the modeling and control groups, fixed in 10% buffered formalin, and decalcified in 5% EDTA. Subsequently, the joints were embedded in paraffin, sectioned, and stained with hematoxylin and eosin Y (H&E) and Safranin O-fast green staining. Staining was performed according to standard protocols, and histomorphometric data were analyzed by Osteometry XP (OsteoMetrics, Inc.). The evaluation included synovial inflammation and cartilage erosion.

[0053] Synovial inflammation was scored according to H&E staining: 0 = no inflammation, 1 = mild thickening of the synovium or slight infiltration of some inflammatory cells, 2 = mild thickening of the synovium plus slight infiltration of some inflammatory cells, 3 = thickening of the synovium, presence of inflammatory cells in the synovial space, 4 = high infiltration of the synovium with many inflammatory cells.

[0054] Cartilage destruction was scored according to Safranin O-fast green staining (0-4): 0 = no destruction, 1 = minimal erosion limited to a single point, 2 = mild to moderate erosion in a limited area, 3 = more extensive area of erosion, 4 = general destruction and destruction of the joint structure.

[0055] The results showed that the SIRT3 knockout mouse model of arthritis induced more obvious inflammatory cell infiltration in the knee joint of the mouse and more obvious destruction of the articular cartilage than the wild-type mouse model induced in the same way (Fig. 3). Figures 4-7 ).

[0056] Joint CT scanning: Bone mass and structure were evaluated by micro-computed tomography, and the knee joint and connected femur, tibia, and fibula tissues of the mice were fixed and scanned using a Skyscan 1076 in vivo μCT scanner (Bruker). The structure parameters such as bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number per unit length (Tb.N) were analyzed on the three-dimensional images obtained by CT An (Skyscan). The results showed that the SIRT3 knockout mouse model of arthritis induced more obvious destruction of the knee joint cartilage and more obvious decrease in bone density than the wild-type mouse model induced in the same way (Fig. 4). Figures 8-10 ).

[0057] HE staining and Safranin O-fast green staining after CD4+ T cell depletion: CD4+ T cell depletion experiments were performed according to previous literature. Briefly, rats were given anti-mouse CD4 antibody (500 pg per mouse; clone GK1.5, Bio X cell) or rat IgG2b isotype control (500 pg per mouse, Bio X cell) intraperitoneally one day before the first immunization (day 0). Then the mice were induced for AIA model, while the CD4 antibody and control antibody were injected every three days for two weeks, until the end of the AIA induction model. At the end of the model, the samples were taken for HE staining and Safranin O-fast green staining, as described above. The results showed that the SIRT3 deletion-induced rheumatoid arthritis phenotype (knee joint destruction, inflammatory cell infiltration) disappeared after CD4+ T cell depletion in mice, indicating that SIRT3 mainly acts through mouse CD4+ T cells Figure 11 , 12).

[0058] To confirm whether the CD4+ T cells were completely depleted by the antibody, blood cells were collected on day 1 and day 14 of the experiment. The blood was lysed, and then stained with anti-mouse CD45 Phycoerythrin (PE, Biolegend), CD3-PerCPCy5.5 (Biolegend), and CD4-Fluorescein Isothiocyanate (FITC, Biolegend). All samples were evaluated using a BD flow cytometer (celesta) and analyzed using FlowJo version 7.6.5 software (Tree Star, Ashland, OR, USA). Spleen and lymph node cells were also lysed and stained with CD45, CD3, and CD4. The results showed that the CD4+ T cells in mice treated with CD4 depletion antibodies decreased or even disappeared Figure 13 ).

[0059] Multi-factor detection: T cell-related inflammatory factors were detected using the Th1 / Th2 / Th17 Cytokine Kit (BD) according to the manufacturer's instructions. The experiment was performed according to the instructions. The optical density was read at 450 nm on a microplate reader (M1000, TECAN). The results showed that the inflammatory factors in the SIRT3 knockout mouse arthritis induction model were significantly higher than in the wild-type mouse model induced in the same way Figure 14 ).

[0060] Flow cytometry: CD4+ naive T cells were sorted using the Miltenyi kit, stimulated with CD3 / CD28 activation antibodies for 72 hours, and the ratio of apoptotic T cells was measured by 7AAD and PE Annexin V (annexin V) using a flow cytometer from BD. The results showed that knocking down SIRT3 in human CD4+ T cells significantly increased T cell apoptosis Figure 15, 16).

[0061] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the technical principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for constructing an animal model of rheumatoid arthritis, characterized by, The SIRT3 knockout mouse in C57BL / 6 background is used to model rheumatoid arthritis induced by mBSA antigen, wherein the modeling of the rheumatoid arthritis induced by mBSA antigen comprises: 1) Primary immunization: on day 1, methylated bovine serum albumin mBSA is dissolved in PBS or normal saline, filtered after dissolution, and configured into a solution of 2 μg / μl; the concentration of complete Freund's adjuvant CFA is 5 mg / ml; the prepared mBSA is mixed with CFA at a ratio of 1:1, and the final concentration of mBSA is 1 μg / μl; the mixture is injected into the root of the mouse tail, 200 μl is injected into the root of the mouse tail for each mouse, and the final total dose of mBSA injected into each mouse is 200 μg; 2) Secondary immunization: on day 7, the mouse knee joint is injected with mBSA, and the mBSA is dissolved in PBS or normal saline, filtered after dissolution, and configured into a solution of 20 μg / μl; 10 μl is injected; the needle is inserted from the lateral side of the knee joint and injected into the knee joint space; the degree of movement of the mouse can be observed after the secondary immunization, and the observation is performed for 7 days; on day 14, the modeling is completed, the knee joint of the mouse is examined by imaging, the knee joint is taken, fixed, sectioned and dyed, and the knee joint damage and inflammatory infiltration of the mouse can be observed. The SIRT3 knockout mouse used for the antigen-induced rheumatoid arthritis model is a female mouse after 8 weeks of adulthood.

2. The construction method of claim 1, wherein, When the background of the SIRT3 knockout mouse is not C57BL / 6 background, the SIRT3 knockout mouse is continuously backcrossed with C57BL / 6 strain mice for more than 10 generations, so that the SIRT3 knockout mouse becomes C57BL / 6 background.

3. The construction method of claim 1, wherein, ​

Images