动物造模,药效评价,慢性结肠炎 z-bio 2024-07-01 385

　　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 (FITC) labeled anti mouse CD45RB monoclonal antibody, phycoerythrin (PE) labeled anti mouse CIM monoclonal antibody, FITC labeled anti mouse CD25 monoclonal antibody, and 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 isolate 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. After 30 minutes, wash twice with PBS to remove unbound antibodies. Then, CD45 RBhigh CD4+immature T cells were isolated and obtained using flow cytometry (FACS Vantage), and their purity was analyzed to be ≥ 98%.

　　(2) Intracellular transplantation: Adjust the concentration of CD45RBhigh CD4+immature T cells and inject them into mice with severe combined immunodeficiency disease (SCID) of the same genotype via intraperitoneal or tail vein injection at a rate of 5 × 100000 cells per mouse (200 μ l PBS solution), and continue feeding.

　　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 softer stools at 3-5 (4.1 ± 0.3) weeks; From 6 to 8 weeks (7.4 ± 0.6), obvious symptoms of chronic colitis appear, including significant weight loss, diarrhea, mucopurulent and bloody stools, prolapse, upright hair, and loss of luster.

　　5. Pathological and biochemical changes after modeling: The model group mice showed symptomatic thickening of the colitis and thickening of the intestinal wall. Mainly consisting of 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 submucosal layer, with the formation of inflammatory granulomas. Ulcers form on the intestinal epithelium, with 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 branching 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, and under the microscope, there is obvious lymphocyte infiltration and inflammatory necrosis of some liver cells in the portal area of the liver. Occasionally, a small amount of lobulated nuclear giant cells can be found in the portal area. Mesenteric lymph nodes reappear and enlarge.

　　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 ± 8.5)%]; The secretion of IFN - γ, IL-2, and TNF - α by CD4+T cells in intestinal mucosal tissue increased compared to the control group, while the secretion of IL-4 and IL-5 remained unchanged.