动物造模,药效评价,诱发溃疡性结肠炎 z-bio 2024-08-21 443

　　(1) Method of replication: Adult SD rats, 5-6 months old, weighing 250-400g, were implanted with a colon of 3-4cm under the right renal capsule of 14-16-day-old homologous fetal rats. On postoperative days 7, 14, and 21, 5-10ml of cardiac blood was extracted for the separation of cardiac blood lymphocytes (HBL). At the same time, the host colon and fetal mouse colon were taken out, partially dehydrated, embedded, and cut into 6 μ m thick pathological sections. After HE staining, they were observed under an optical microscope, and partially used for the separation of colonic mucosal lymphocytes (CML) and colonic epithelial cells (CEC). On this basis, the cytotoxic activity of host mouse HBL and CML against CEC was determined. HBL, CML, and CEC separation methods: Heparin treated cardiac blood mononuclear cells were separated using Ficoll Hypaque gradient separation method, monocytes and macrophages were removed using glass adhesion method, and the lymphocyte concentration was adjusted to 1 × 1000000/ml using TcA solution (199Tc, composed of 10% fetal bovine serum, 100U/ml penicillin, and 100U/ml streptomycin, etc.), with survival cells above 95%. CEC was separated using Keoder's method, with the latter concentration adjusted to 1 × 1000000000/ml, and survival epithelial cells above 89%. Cytotoxicity assay method: Add 150 μ l/well of CEC (target cell) with a concentration of 1 × 100000/ml to the micro assay plate, and culture for 12 hours in an environment of 37 ℃, 5% CO2, and 95% O2. After removing the supernatant, add 150 μ l/well of HBL and CML (effector cells), with a ratio of 100:1 effector cells to target cells. Only TcA culture medium was added to the spontaneously removed target cell wells, and double distilled water was added to the maximum removed target cell wells. All samples were made into 3 portions, with a total culture volume of 0.3ml and a pH value adjusted to 7.2. The culture was continued for 12 hours at 37 ℃. Finally, the CEC that did not adhere to the micro assay plate wall was removed using Hank's solution, leaving the CEC that adhered to the wall. The cytotoxicity index was calculated under a phase contrast microscope using the following formula. Cytotoxicity index (%)=[(target cells removed experimentally - spontaneously removed target cells)/(maximum removed target cells - spontaneously removed target cells)] × 100%.

　　(2) 7 days after surgery, the animal fetal colon began to have inflammatory cell infiltration, some mucosal glands and epithelial cells shed, and the mucosa became thinner. There is a large amount of mucus in the intestinal lumen of the transplant, which compresses the tissue and causes the formation of a cystic cavity. At 14 days, the above changes became more severe, with all mucous membranes damaged, the number of glands reduced, a large amount of necrotic material in the intestinal lumen, and lymphocyte and monocyte infiltration extending from the mucosal muscle layer to the serosal layer. At 21 days, the mucosal glands severely atrophied and degenerated, with fibrous connective tissue hyperplasia. In vitro cytotoxicity assays showed that the cytotoxicity indices of HBL and MCL in model animals were significantly higher than those in normal control mice.

　　(3) The colon of fetal mice implanted in comparative medicine is an immunogen for adult mice of the same lineage, which can cause a series of immune responses in host mice. This model shows that when the colon of fetal mice is implanted under the renal capsule of adult homologous mice, the former experiences mucosal necrosis and shedding, inflammatory cell infiltration, and presents a typical host anti graft response; At the same time, there is also infiltration of inflammatory cells in the colonic lamina propria of host mice, characterized by lymphoid follicles and sometimes mucosal erosion and ulceration. There are positive antibodies against implanted fetal mouse colon in the serum of host mice, as well as positive antibodies against self colon in the serum, indicating the presence of cross antigens between the host colon and implanted fetal mouse colon. In vitro cytotoxicity assays showed that host mouse cardiac blood lymphocytes (HBL) and colon mucosal lymphocytes (CML) significantly increased their cytotoxic activity against their own colonic epithelial cells (CEC), indicating that antibody dependent cell-mediated cytotoxicity (ADCC) plays an important role in the replication process of this model, suggesting that the cytotoxicity process of ulcerative colitis also requires the mediation of antibodies or immune complexes. Given the above characteristics, this model has good application value in the field of medical basic research as an experimental tool for exploring the etiology and pathogenesis of ulcerative colitis. However, due to the fact that fetal mouse colon transplantation is the core technology of this model, the technical requirements are high, the experimental period is long, and the success rate is relatively low.