An improved method for an ANCA-associated vasculitis mouse model induced by anti-GBM antibody

Abstract

This invention discloses a method for improving a mouse model of ANCA-associated vasculitis induced by anti-GBM antibodies, relating to the field of animal model construction. The method involves subcutaneously injecting mice with 20 μg rMPO on Day 0; administering 10 μg rMPO on Day 7; subcutaneously injecting 250 μg / kg GCSF daily from Day 11 to 15; and injecting 100 μl of goat anti-GBM serum via the tail vein on Day 16 to obtain the improved mouse model. This method improves the existing anti-GBM antibody-induced ANCA-associated vasculitis mouse model by injecting GCSF. This model enhances the disease severity in ANCA-associated vasculitis mice, specifically reflecting the disease in renal pathology and proteinuria, making the differences more obvious and effectively improving the success rate of model establishment.

Description

Technical Field

[0001] This invention relates to animal model construction technology, specifically to a method for improving a mouse model of ANCA-associated vasculitis induced by anti-GBM antibody. Background Technology

[0002] Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by vascular inflammation, endothelial damage, and tissue injury. Recent data show that the prevalence of AAV is 300–421 per million people, suggesting that this disease is not rare. Its serological characteristics are typically myeloperoxidase (MPO)-ANCA positivity or proteasome 3 (PR3)-ANCA positivity.

[0003] There are five existing classic mouse models of ANCA-associated vasculitis, one of which is an anti-GBM antibody-induced model. When we replicated this method in China, we found that the experimental group mice did not show any difference from the control group in terms of modeling results. There were no significant differences between the model group mice and the control group mice in terms of kidney pathology and urinary protein, which reflect the severity of the disease. Therefore, it is necessary to improve the modeling method for this mouse model.

[0004] This approach was designed to improve the model establishment rate—by injecting granulocyte colony-stimulating factor (GCSF) into the original modeling method to enhance the model. Results showed that the model group mice differed significantly from the control group mice in renal pathology and urinary protein, reflecting disease severity, effectively improving the model establishment success rate. Summary of the Invention

[0005] The purpose of this invention is to provide an improved method for an ANCA-associated vasculitis mouse model induced by anti-glomerular basement membrane (GBM) antibodies, in order to solve the problem that there is no significant difference between the model mice constructed according to the existing technology and the control mice in terms of kidney pathology, urinary protein, etc., which reflect the severity of the disease.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for improving a mouse model of ANCA-related vasculitis induced by anti-GBM antibody, comprising the following steps:

[0007] Mice were subcutaneously injected with 20 μg rMPO dissolved in Freund's complete adjuvant at Day 0.

[0008] Administer 10 μg rMPO dissolved in Freund's incomplete adjuvant on Day 7;

[0009] GCSF was administered subcutaneously at a dose of 250 μg / kg daily for 5 consecutive days from Day 11 to 15, with the specific dosage calculated based on body weight.

[0010] On Day 16, 100 μl of goat anti-mouse GBM serum was injected via the tail vein to obtain a modified mouse model.

[0011] Preferably, the modified mouse model is used for 4 days after it matures.

[0012] Preferably, the mouse is a 6-8 week old C57 male mouse.

[0013] Compared with the prior art, the present invention provides a method for improving the mouse model of ANCA-related vasculitis induced by anti-GBM antibody. The original anti-GBM antibody-induced ANCA-related vasculitis mouse model is improved by injecting GCSF, which enhances the differences between the model group mice and the control group mice in terms of kidney pathology, urinary protein, etc., which reflect the severity of the disease, making the differences more obvious and effectively improving the success rate of model establishment. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0015] Figure 1 This is a flowchart of the improved mouse modeling method provided in an embodiment of the present invention;

[0016] Figure 2 This is a comparison chart of the modeling results of two groups of mice that received GCSF stimulation, provided as an embodiment of the present invention. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0018] As attached Figure 1 To be continued Figure 2 As shown:

[0019] Example:

[0020] This invention provides a method for improving a mouse model of ANCA-related vasculitis induced by anti-GBM antibody, comprising the following steps:

[0021] Mice were subcutaneously injected with 20 μg rMPO dissolved in Freund's complete adjuvant at Day 0.

[0022] Administer 10 μg rMPO dissolved in Freund's incomplete adjuvant on Day 7;

[0023] GCSF was administered subcutaneously at a dose of 250 μg / kg daily for 5 consecutive days from Day 11 to 15, with the specific dosage calculated based on body weight.

[0024] On Day 16, 100 μl of goat anti-mouse GBM serum was injected via the tail vein to obtain a modified mouse model. This model could be used for 4 days after it matured. The mice were then euthanized on Day 20.

[0025] As shown above, this method improves the existing ANCA-associated vasculitis mouse model by injecting GCSF, thereby aggravating the disease severity in ANCA-associated vasculitis mice and specifically reflecting the differences in kidney pathology and urinary protein, thus significantly improving the success rate of model establishment.

[0026] The modified mouse model is used for 4 days after it matures.

[0027] The agents used in this method are rMPO (laboratory-synthesized MPO used to stimulate MPO-ANCA production), Freund's complete adjuvant, Freund's incomplete adjuvant, GCSF (commercial granulocyte colony-stimulating factor), and anti-GBM serum (PROBETEX, PTX-001AGBM).

[0028] The mice were 6-8 weeks old C57 males.

[0029] Reference Figure 2 This figure compares the results of two groups of mice that received GCSF stimulation, including results on mouse kidney pathology, urinary protein, and serum creatinine. Figure captions: a. Increased severity of proteinuria in mice after GCSF administration; b. Significantly increased serum creatinine in mice after GCSF administration; c. Differences in MPO-ANCA antibody titers between groups before and after GCSF administration (GCSF had no significant effect on antibody titers); d. Pathological results indicate that although the proportion of abnormal glomeruli differed between the two groups before and after GCSF administration, the proportion of abnormal glomeruli significantly increased after GCSF administration; e. Typical pathological images for each group: NA-OVA normal glomeruli, NA-MPO necrotic glomeruli, GCSF-OVA normal glomeruli, and GCSF-MPO glomeruli after crescent formation.

[0030] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for improving a mouse model of ANCA-associated vasculitis induced by anti-GBM antibodies, the method comprising the steps of Comprise: On Day 0, the mice were injected subcutaneously with 20 μg rMPO dissolved in Freund's complete adjuvant; On Day 7, the mice were injected subcutaneously with 10 μg rMPO dissolved in Freund's incomplete adjuvant; On Day 11-15, the mice were injected subcutaneously with 250 μg / kg GCSF every day for 5 consecutive days, and the amount of the drug injected was calculated according to the body weight; On Day 16, the mice were injected intravenously with 100 μl goat anti-mouse GBM serum to obtain a modified mouse model.

2. The method for improving the mouse model of ANCA-associated vasculitis induced by anti-GBM antibody according to claim 1, wherein, The modified mouse model has a service life of 4 days after maturation.

3. The method for improving the mouse model of ANCA-associated vasculitis induced by anti-GBM antibody according to claim 1, wherein, The mice are 6-8 weeks old C57 male mice.

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