动物造模,慢性结肠炎模型 z-bio 2023-10-09 770

　　1. Animal modeling materials: Female BALB/c mice (H-2 gene phenotype), aged 8-10 weeks at the time of use; Reagents and antibodies: Hamster anti mouse CD3 monoclonal antibody, fluorescein isothiocyanate labeled anti mouse CD45RB monoclonal antibody, phycoerythrin labeled anti mouse CIM monoclonal antibody, FITC labeled anti mouse CD25 monoclonal antibody, FITC labeled anti mouse CD69 monoclonal antibody, RPMI1640 culture medium, fetal bovine serum, penicillin, streptomycin, levoglutamine, and gentamicin.

　　2. Modeling method

　　(1) Isolation of C1345RBhigh CD4+immature T cells: Firstly, use Dynal magnetic beads to separate CD4+T cells from the spleen of healthy BALB/c mice, and strictly follow the instructions for specific operations. Incubate FITC labeled CD45RB monoclonal antibody and PE labeled CD4 monoclonal antibody with CD4+T cells, and after 30 minutes, wash twice with PBS to remove unbound antibodies. Then, CD45 RBhigh CD4+immature T cells were separated and obtained using flow cytometry, and their purity was analyzed to be ≥ 98%.

　　(2) In vivo cell transplantation: Adjust the CD45RBhigh CD4+immature T cell concentration by pressing 5 × 100000 pieces/piece (200 μ PBS solution was injected into mice with severe combined immunodeficiency disease of the same genotype through the abdominal cavity or tail vein, and continued to be raised.

　　3. Modeling principle: CD45RBhigh CD4+immature T cells can induce chronic colitis in SCID mice.

　　4. General changes after modeling: The model group mice showed weight loss and softened stools at 3-5 (4.1 ± 0.3) weeks; From 6 to 8 weeks (7.4 ± 0.6), obvious symptoms of chronic colitis appear, characterized by significant weight loss, diarrhea, mucopurulent and bloody stools, anal prolapse, upright hair, and loss of luster.

　　5. Pathological and biochemical changes after modeling: The model group mice showed colitis thickening and intestinal wall thickening. Mainly ascending colon and transverse colon. Under the microscope, the entire colon wall is infiltrated with a large number of T lymphocytes, monocytes and macrophages, as well as a small amount of neutrophils and eosinophils, especially in the mucosal layer and submucosa, with the formation of inflammatory granulomas. Ulcer formation in the intestinal epithelium, loss of mucus on the surface of epithelial cells, disappearance of goblet cells, and abscess of glandular fossa. Gland hyperplasia, elongation, surface structural deformation of villi, and bifurcation like changes. In addition, some animals also showed significant inflammatory changes in the cecum, distal ileum, and stomach. The spleen and liver both increase to varying degrees. Microscopically, there is significant lymphocyte infiltration and inflammatory necrosis of some liver cells in the portal area of the liver. Occasionally, a small number of lobulated nuclear giant cells can be found in the portal area. The mesenteric lymph nodes reappear and increase.

　　Through flow cytometry analysis, it was found that the surface expression of CD4+T cells in the lamina propria of the inflamed colon mucosa was quite high, with CD69 [(72.5 ± 12.5)%] and CD25 [(32.5)%]