Rheumatoid arthritis mouse model construction method and mouse model

Abstract

The invention discloses a construction method of a rheumatoid arthritis mouse model. The construction method comprises the following steps: S1, preparing a type II collagen emulsion; s2, preparing an antibody mixture; s3, dissolving the LPS in the sterile PBS (Phosphate Buffer Solution); s4, injecting the II-type collagen emulsion into the subcutaneous part of the tail root of the mouse; s5, intravenously injecting the antibody mixture into the tail of the mouse on the 21st day; s6, injecting LPS into the abdomen of the mouse on the 22th day to obtain the rheumatoid arthritis mouse model; according to the construction method and the mouse model, the mechanism that inherent immunity and adaptive immunity are combined to promote the development of arthritis is combined, compared with traditional C IA, the morbidity and morbidity are remarkably improved, the modeling time is short, compared with traditional CAIA, the morbidity and morbidity index are equivalent, various types of autoantibodies can be induced to be generated, meanwhile, cost is reduced, and the mouse model has good application prospects. The joint pathological change is closer to rheumatoid arthritis, the success rate of the model is improved, and the repeatability and scientificity of the model are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction. Specifically, it is a method for constructing a rheumatoid arthritis mouse model and the mouse model. Background Art

[0002] Currently, there are two recognized existing solutions for constructing an RA animal model in C57 / B6 mice: one is to directly use the CIA model for subcutaneous / intradermal injection at the root of the tail of C57 / B6 mice. This model construction period is very long, generally at least 8 weeks, with a low incidence rate, and most reports show an incidence rate <50%. However, the simulation of joint inflammation and bone erosion is better; the other method uses the Collagen Antibody Induced Arthritis model (CAIA model). After injecting collagen antibody subcutaneously / intradermally at the root of the tail of C57 / B6 mice, it can develop disease 3 days after injection and reach the peak 10 days later, with a relatively fast onset. However, the incidence rates of joint inflammation, bone erosion, etc. are low, and the cost is expensive. There is currently no relevant report on how to construct an RA animal model by combining the advantages of the two models in C57 / B6 mice. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing a rheumatoid arthritis mouse model and the mouse model, which combines innate immunity and adaptive immunity to promote the development mechanism of arthritis. Compared with the traditional CIA, the incidence rate and morbidity are significantly improved, and the modeling time is short. Compared with the traditional CAIA, the incidence rate and morbidity index are equivalent, various types of autoantibodies can be induced, and at the same time, the cost is reduced, making the joint pathological changes closer to rheumatoid arthritis, improving the model success rate, and ensuring the repeatability and scientificity of the model.

[0004] To solve the above technical problem, the present invention provides the following technical solutions:

[0005] A method for constructing a rheumatoid arthritis mouse model, comprising the following steps:

[0006] S1. Mix chicken type II collagen and an equal volume of complete Freund's adjuvant to obtain a type II collagen emulsion;

[0007] S2. Dissolve a mixture of five antibodies, namely clone A2-10, F10-21, D8-6, D1-2G, and D2-112, in sterile PBS to obtain an antibody mixture;

[0008] S3. Dissolve LPS in sterile PBS;

[0009] S4. Inject the type II collagen emulsion subcutaneously at the root of the mouse tail to cause inflammation in the mouse;

[0010] S5. On the 21st day after inflammation is induced, inject the antibody mixture into the tail vein of the mice in S4.

[0011] S6. Inject LPS into the abdomen of the mice in S5 on the 22nd day, and a rheumatoid arthritis mouse model can be obtained after 35 days.

[0012] Furthermore, in step S1, chicken type II collagen is dissolved in 0.05 M glacial acetic acid, and the concentration of chicken type II collagen after dissolution is 4 mg / mL.

[0013] The concentration of complete Freund's adjuvant is 4 mg / mL, and the concentration of type II collagen emulsion is 1 mg / mL.

[0014] Furthermore, in step S2, the concentration of the antibody mixture is 10 mg / mL.

[0015] Furthermore, in step S3, the concentration of LPS is 500 μg / mL, and the pH of LPS is 7.4.

[0016] Furthermore, in step S4, the injection volume of the type II collagen emulsion per single mouse is 0.1 mL / mouse.

[0017] Furthermore, in step S5, the injection volume of the antibody mixture per single mouse is 0.4 - 1 mg / mouse.

[0018] Furthermore, in step S7, the injection volume of LPS per single mouse is 50 μg / mouse.

[0019] Furthermore, the strain of the mice is C57BL / 6.

[0020] A rheumatoid arthritis mouse model prepared by a method for constructing a rheumatoid arthritis mouse model.

[0021] The technical solution of the present invention has achieved the following beneficial technical effects:

[0022] The present application provides a method for constructing a rheumatoid arthritis mouse model and a mouse model, which combines the mechanisms of innate immunity and adaptive immunity to promote the development of arthritis. Compared with traditional CIA, the incidence rate and morbidity are significantly increased, and the modeling time is short. Compared with traditional CAIA, the incidence rate and morbidity index are comparable, various types of autoantibodies can be induced, and at the same time, the cost is reduced, making the joint pathological changes closer to rheumatoid arthritis, improving the model success rate, and ensuring the repeatability and scientificity of the model. Brief Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the method for constructing a rheumatoid arthritis mouse model and the model of the present invention;

[0024] Among them, A is a schematic diagram of the construction process of a mouse model of rheumatoid arthritis;

[0025] B is the analysis of the incidence rate (left) and clinical score (right) of the present invention and other existing mouse arthritis models;

[0026] C are representative photos of the hind paw ankle joints of mice in each group, micro-CT images, hematoxylin-eosin staining (H&E), safranin O-fast green cartilage staining (SO / FG), and tartrate-resistant acid phosphatase staining (TRAP) typical pictures;

[0027] D is the histological quantitative analysis of micro-CT data;

[0028] E is the quantitative analysis of joint inflammation and pathological changes;

[0029] F is the analysis of the production of anti-type II collagen antibodies in the sera of mice in each group. Specific implementation mode

[0030] A method for constructing a mouse model of rheumatoid arthritis, comprising the following steps:

[0031] S1. Mix chicken type II collagen and an equal volume of complete Freund's adjuvant to obtain a type II collagen emulsion;

[0032] S2. Dissolve a mixture of five antibodies, clone A2-10, F10-21, D8-6, D1-2G, and D2-112, in sterile PBS to obtain an antibody mixture;

[0033] S3. Dissolve LPS in sterile PBS;

[0034] S4. Inject the type II collagen emulsion subcutaneously at the base of the mouse tail to cause inflammation in the mouse;

[0035] S5. On the 21st day after the inflammation is induced, inject the antibody mixture into the tail vein of the mouse in S4;

[0036] S6. Inject LPS into the abdomen of the mouse in S5 on the 22nd day, and a mouse model of rheumatoid arthritis can be obtained after 35 days.

[0037] Example 1

[0038] 1. Experimental animals: C57BL / 6.

[0039] 2. Experimental grouping: including CIA group, CAIA group, C&AIA group, antibody control group, and non-modeling control group.

[0040] 3. Construction of CIA model:

[0041] Reagents: Complete Freund's adjuvant CFA containing Mycobacterium tuberculosis (its concentration is related to the induced anti-collagen antibody), 4 mg / ml; in 10 mg of lyophilized chicken type II collagen powder, 2.5 ml of 0.05 M glacial acetic acid was added and it automatically dissolved overnight, with a final concentration of 4 mg / ml.

[0042] First, connect two 5-ml syringes without black rubber with a rubber tube. Pull out the syringe barrel of one of them. First add glacial acetic acid with a pipette, then add collagen, and finally add CFA. Dissolve chicken type II collagen in 0.05 M glacial acetic acid at a ratio of 1:1 (28.6 μl of glacial acetic acid was dissolved in 10 ml of ddH2O), and then dissolve it in CFA for emulsification. Use an emulsifier to emulsify on ice for 30 min (2100 times) to prepare a type II collagen emulsion, with a final concentration of the type II collagen emulsion of 1 mg / ml.

[0043] Treatment of the CIA group:

[0044] On Day 0, for the primary immunization, inject 0.1 mL of the type II collagen emulsion subcutaneously at the base of the mouse tail to cause inflammation in the mouse.

[0045] On Day 21, for the second immunization, inject 0.1 mL of the type II collagen emulsion subcutaneously again at the base of the small tail as a booster injection.

[0046] From Day 36 to 56, the mice will show joint swelling, which develops from the hind feet to the front feet and then to the tail.

[0047] 4. Construction of the CAIA model

[0048] Reagents for CAIA preparation:

[0049] Antibody mixture: Dissolve a mixture of five antibodies, clone A2-10 (IgG2a), F10-21 (IgG2a), D8-6 (IgG2a), D1-2G (IgG2b), and D2-112 (IgG2b), or the mixed antibodies (D1, F10, A2, D8), 10 mg each in 4 ml of sterile PBS, with a final concentration of 10 mg / ml. The recommended dose for C57BL / 6 by Chondrex is 5 mg / mice.

[0050] Dissolve 500 μg of LPS in 1 ml of sterile PBS, with a final concentration of 500 μg / ml and a pH of 7.4.

[0051] Treatment of the CAIA group:

[0052] On Day 21, inject the antibody mixture intravenously at a dose of 4 mg / mouse into the tail vein.

[0053] On Day 22, inject LPS at a dose of 50 μg / mouse into the peritoneal cavity of the mouse.

[0054] If the mice have difficulty walking or eating, analgesic Temgesic at a dose of 0.1 mg / kg in 50 μl should be injected subcutaneously.

[0055] 5. Treatment of the C&AIA group:

[0056] On Day 0, for the primary immunization, 0.1 mL of type II collagen emulsion was injected subcutaneously at the base of the mouse tail to induce inflammation in the mice.

[0057] On Day 21, the antibody mixture was injected via the tail vein at a dose of 1 mg / mouse.

[0058] On Day 22, LPS was injected into the peritoneal cavity of the mice at a dose of 50 μg / mouse.

[0059] Arthritis developed from Day 27 to 35, and reached its maximum from Day 31 to 35.

[0060] 6. Treatment of the antibody control group:

[0061] On Day 21, the antibody mixture was injected via the tail vein at a dose of 1 mg / mouse.

[0062] On Day 22, LPS was injected into the peritoneal cavity of the mice at a dose of 50 μg / mouse.

[0063] 7. Treatment of the non-modeled control group:

[0064] On Day 21, the antibody mixture was injected via the tail vein at a dose of 1 mg / mouse.

[0065] To quantify the degree of inflammation in each group, the arthritis development in mice was divided into four grades, and the scoring criteria were as follows: Clinical score 0: normal paw; Clinical score 1: slightly red and swollen paw; Clinical score 2: moderately red and swollen paw; Clinical score 3: entire paw red and swollen; Clinical score 4: maximum red and swelling.

[0066] The results are as Figure 1 shown, Figure 1 A is a schematic diagram of the modeling protocol for this model.

[0067] Figure 1 B shows the incidence and arthritis score evaluated daily starting from Day 23. Figure B (left) shows the arthritis incidence in each group of C57BL / 6 mice in the C&AIA group, antibody control, CAIA group, CIA group, and non-modeled group. Figure B (right) shows the clinical score of arthritis in C57BL / 6 mice in the C&AIA group, antibody control, CAIA group, CIA group, and non-modeled group after immunization with chicken type II collagen or injection of antibody complex via the tail vein. After modeling, the change trend of the C&AIA group was similar to that of the CAIA group. The arthritis in the C&AIA group reached its maximum on Day 28 - 29 and remained stable on Day 30 - 31, and the severity of arthritis was significantly higher than that of the other groups.

[0068] Figure 1 C shows representative photographs, micro-computed tomography (CT) images, hematoxylin-eosin (H&E) staining, safranin O-fast green cartilage (SO / FG) staining, and TRAP staining of the hind paw ankle joint. The scale bar is 1 mm (1x) and 200 μm (10x). After C57BL / 6 mice were immunized with chicken type II collagen or injected with antibody complexes via the tail vein, micro-CT scanning was performed on the representative left hind paws of each group of mice. After paraffin sections were stained with hematoxylin-eosin (H&E), safranin O-fast green cartilage (SO / FG), and tartrate-resistant acid phosphatase (TRAP), the typical pathological changes were observed. It was found that in the C&AIA group, synovial hyperplasia with inflammatory cell infiltration was more severe, there was loss of cartilage matrix, delamination damage, more bone erosion and osteophyte formation, and activated osteoclasts were seen by TRAP staining, with enhanced bone resorption, presenting typical pathological features of rheumatoid arthritis overall.

[0069] Figure 1 D shows the histomorphometric analysis of micro-CT data. According to the micro-CT scan results, the total bone volume (TV), bone volume (BV), bone surface area (BS), bone volume fraction (BV / TV), ratio of bone surface area to total volume (BS / TV), ratio of bone surface area to bone volume (BS / BV), trabecular bone spacing (Tb.Sp), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and bone mineral density (BMD) of each group of mice were quantitatively compared. The results showed that the bone damage induced by the C&AIA group was similar to that of the CAIA group, with more significant bone mass reduction (decrease in BV / TV) and trabecular bone rarefaction (increase in Tb.Sp and Tb.N) in the C&AIA model, indicating that the degree of bone destruction was more severe than that in the CIA group.

[0070] Figure 1 E shows the quantitative analysis of pathological tissue sections. The results showed that severe inflammatory cell infiltration and bone erosion occurred in the C&AIA group of mice. Pathological scores (including synovitis, pannus formation, marginal erosion, structural changes, and total score) indicated that the degree of joint injury and inflammation was comparable to that of the CAIA group, and both were much higher than that of the CIA model, indicating that the C&AIA model performed no worse than the CAIA model in terms of inflammatory scores.

[0071] Figure 1F shows the production of anti-type II collagen antibodies in the sera of C57BL / 6 mice after immunization with chicken type II collagen or intravenous injection of antibody complexes. The sera of mice in the CIA group were taken on Day 56, and the sera of the remaining mice were taken from mice on Day 32. The results showed that among the mice in each group, the levels of endogenous anti-collagen type II (CⅡ) antibodies (including IgA, IgM, IgG1, and IgG3) in the sera of mice in the C&AIA group were the highest, indicating that the advantage of the C&AIA model in humoral immunity is that it can induce a faster antibody response and higher antibody levels, significantly shortening the experimental period and enhancing the sensitivity of the model.

[0072] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to exhaustively list all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the claims of this patent application.

Claims

1. A method for constructing a mouse model of rheumatoid arthritis, characterized in that, It includes the following steps: S1. Mix chicken type II collagen with an equal volume of complete Freund's adjuvant to prepare a type II collagen emulsion; S2. Dissolve a mixture of five antibodies, namely clone A2-10, F10-21, D8-6, D1-2G, and D2-112, in sterile PBS to prepare an antibody mixture; S3. Dissolve LPS in sterile PBS; S4. Inject the type II collagen emulsion subcutaneously at the root of the mouse tail to induce inflammation in the mouse; S5. On the 21st day after inflammation is induced, inject the antibody mixture into the tail vein of the mouse in S4; S6. On the 22nd day, inject LPS into the abdomen of the mouse in S5, and a rheumatoid arthritis mouse model can be obtained after 35 days.

2. The method for constructing a rheumatoid arthritis mouse model according to claim 1, wherein In step S1, chicken type II collagen is dissolved in 0.05M glacial acetic acid, and the concentration of chicken type II collagen after dissolution is 4mg / mL; The concentration of complete Freund's adjuvant is 4mg / mL, and the concentration of the type II collagen emulsion is 1mg / mL.

3. The method for constructing a rheumatoid arthritis mouse model according to claim 1, wherein In step S2, the concentration of the antibody mixture is 10mg / mL.

4. The method for constructing a rheumatoid arthritis mouse model according to claim 1, characterized in that, In step S3, the concentration of LPS is 500μg / mL, and the pH of LPS is 7.

4.

5. The method for constructing a mouse model of rheumatoid arthritis according to claim 1, characterized in that, In step S4, the injection volume of the type II collagen emulsion per mouse is 0.1mL / mouse.

6. The method for constructing a rheumatoid arthritis mouse model according to claim 1, characterized in that, In step S5, the injection volume of the antibody mixture per mouse is 0.4 - 1mg / mouse.

7. The method for constructing a rheumatoid arthritis mouse model according to claim 1, wherein, In step S7, the injection volume of LPS per mouse is 50μg / mouse.

8. The method for constructing a rheumatoid arthritis mouse model according to claim 1, characterized in that, The mouse strain is C57BL / 6.

9. A rheumatoid arthritis mouse model prepared by the method according to any one of claims 1 - 8.

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