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Method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as hepatic fibrosis and primary biliary cirrhosis animal models

A technology for biliary cirrhosis and IL-2R, applied in the field of medical biology, can solve the problems of low level of portal inflammation and bile duct damage, and no development of liver fibrosis.

Inactive Publication Date: 2014-04-30
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, autoimmune cholangitis in IL-2Rα(- / -) mice was not very severe, with lower levels of portal inflammation and bile duct destruction, and did not develop to the degree of liver fibrosis

Method used

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  • Method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as hepatic fibrosis and primary biliary cirrhosis animal models
  • Method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as hepatic fibrosis and primary biliary cirrhosis animal models
  • Method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as hepatic fibrosis and primary biliary cirrhosis animal models

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Establishment of IL-12p40(- / -)IL-2Rα(- / -) mice

[0037] C57BL / 6J background IL-2Rα(- / -) mice (B6.129S4-I12ratm1Dw) [Willerford, D.M., et al. (1995). Immunity 3(4): 521-530] and IL-12p40(- / -) mice (B6.129S1-I112btm1Jm) [Magram, J., et al. (1996). Immunity 4 (5): 471-481] were purchased from Jackson Laboratory, USA (The Jackson Laboratory, Maine, USA , http: / / www.jax.org / ), were raised in a special pathogen-free (SPF) environment in the Experimental Animal Center of the University of Science and Technology of China.

[0038] We crossed IL-2Rα(+ / -) mice with IL-12p40(- / -) mice to obtain IL-12p40(+ / -)IL-2Rα(+ / -) mice; 12p40(- / -) mice were crossed to obtain IL-12p40(- / -)IL-2Rα(+ / -) mice. IL-2Rα(- / -) mice used in the experiments were bred from IL-2Rα(+ / -) mice, and IL-12p40(- / -) IL-2Rα(- / -) mice were bred from IL- 12p40(- / -)IL-2Rα(+ / -) mice were bred. Since the mutant genes of IL-12p40 and IL-2Rα both contain a neo gene, the method for identifying the mouse IL...

Embodiment 2

[0039] Example 2. Histopathological examination

[0040] The liver and colon tissue pieces of mice were fixed in 4% neutral formaldehyde for 1-2 days, dehydrated and embedded in paraffin. Paraffin-embedded liver tissue was cut into 4 μm slices, while colon tissue was cut into 6 μm slices. All slices were dewaxed and H&E stained. Hepatic portal inflammation, bile duct destruction, fibrosis, and colitis were scored by pathologists. Liver tissue was stained by Azan to show fibrosis.

[0041] see results figure 1 , figure 2 and Figure 4 . From figure 1 and figure 2 In the HE staining images of IL-12p40(- / -) IL-2Rα(- / -) mice, the degree of portal lymphocyte infiltration in the liver was significantly higher than that of IL-2Rα(- / -) mice , while the degree of colonic inflammation was significantly lower than that of IL-2Rα(- / -) mice. The results of pathological scoring were the same. Figure 4 Azan staining showed that IL-12p40(- / -) IL-2Rα(- / -) mice had more collagen d...

Embodiment 3

[0042] Example 3. Isolation of mononuclear cells in various organs

[0043] The liver of the mouse was taken out, ground with PBS containing 0.2% BSA, filtered through a steel mesh, centrifuged at 650 rpm for 1 min, and the supernatant was taken. Spleen and mesenteric lymph nodes were ground from two slides with PBS / 0.2% BSA and filtered through nylon mesh. Mononuclear cells from the resuspended liver and spleen cell suspensions were subjected to density gradient centrifugation with 40% and 70% Percoll (GE Healthcare) for 20 min to collect cells in the middle layer. Cell counts were performed under a microscope by a hemocytometer.

[0044] see results image 3 . The number of mononuclear cells in the liver of IL-12p40(- / -)IL-2Rα(- / -) mice was significantly higher than that of IL-2Rα(- / -) mice and IL-2Rα(+ / -) mice .

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Abstract

The invention discloses a method for breeding IL-12p40 (- / -) IL-2R alpha (- / -) mice used as novel hepatic fibrosis and primary biliary cirrhosis animal models. The method includes steps of (1), performing cross breeding on IL-2R alpha (+ / -) mice and IL-12p40(- / -) mice to obtain IL-12p40(+ / -) IL-2R alpha (+ / -) mice by means of breeding; (2), performing cross breeding on IL-12p40(- / -) mice and the IL-12p40(+ / -) IL-2R alpha (+ / -) mice obtained in the step (1) to obtain IL-12p40(- / -) IL-2R alpha (+ / -) mice by means of breeding; (3), performing inbreeding on the IL-12p40(- / -) IL-2R alpha (+ / -) mice obtained in the step (2) and obtaining the identified IL-12p40(- / -) IL-2R alpha (- / -) mice. The method has the advantages that IL-12p40 is an important cell factor in the immune system of a patient, and functions and differentiation of CD4+T and CD8+T cells can be affected by the IL-12p40; IL-2R alpha is an alpha chain of an IL-2 receptor and plays a key role in keeping the balance of the immune system of the patient.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, in particular to a method for cultivating IL-12p40(- / -)IL-2Rα(- / -) mice as animal models of liver fibrosis and primary biliary cirrhosis method. The IL-12p40(- / -)IL-2Rα(- / -) mice obtained by the method of the present invention can be used as a new animal model for screening drugs for treating liver fibrosis and primary biliary cirrhosis. Background technique [0002] Liver fibrosis is a pathological process of excessive accumulation of extracellular matrix proteins, especially collagen, due to chronic liver injury. Liver fibrosis is not an independent disease. Many chronic liver diseases can cause liver fibrosis, including viral hepatitis infection, excessive alcohol consumption, non-alcoholic steatohepatitis, autoimmune hepatitis, etc. In the process of liver fibrosis, hepatic stellate cells are the main effector cells, and the activated hepatic stellate cells synthesize a large amount of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027
Inventor 廉哲雄姚远杨微
Owner UNIV OF SCI & TECH OF CHINA
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