动物造模,药效评价,慢性结肠炎 z-bio 2024-07-29 296

　　1. Modeling material animals: 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, FITC labeled anti mouse CD69 monoclonal antibody, RPMI1640 culture medium, fetal bovine serum, penicillin, streptomycin, L-glutamine, and gentamicin.

　　2. Modeling method

　　(1) C1345RBhigh CD4+immature T cell isolation: First, 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 a flow cytometer (FACS Vantage), and their purity was analyzed to be ≥ 98%.

　　(2) Cell transplantation in vivo: Adjust the concentration of CD45RBhigh CD4+immature T cells and inject them into severe combined immunodeficiency disease (SCID) mice 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 stool softening at 3-5 (4.1 ± 0.3) weeks; At 6-8 weeks (7.4 ± 0.6), obvious symptoms of chronic colitis appeared, manifested as significant weight loss, diarrhea, mucous purulent stools, prolapse, upright hair and loss of luster.

　　5. Pathological and biochemical changes after modeling: In the model group, colitis induced thickening and intestinal wall thickening were observed. Mainly in the ascending colon and transverse colon. Under the microscope, the entire colon wall is infiltrated with a large number of T lymphocytes, mononuclear macrophages, as well as a small number of neutrophils and eosinophils, especially in the mucosal and submucosal layers, and inflammatory granulomas are formed. Ulcer formation in the intestinal epithelium, loss of mucus on the surface of epithelial cells, disappearance of goblet cells, and abscess in the glandular fossa. Glandular hyperplasia, elongation, deformation of villous surface structure, and branching like changes. In addition, some animals also showed significant inflammatory changes in the cecum, terminal ileum, and stomach. The spleen and liver have increased 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 number of lobulated 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 CD69 [(72.5 ± 12.5)%] and CD25 [(32.5 ± 8.5)%] was significantly high on CD4+T cells in the lamina propria of inflamed colon mucosa; The secretion levels 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 levels of IL-4 and IL-5 remained unchanged.