动物造模,溃疡性结肠炎动物模型 z-bio 2021-11-26 749

　　(1) Replication method Adult male BALB/c mice, abdomen (without damage to the skin) about 2.0 cm x 1.5 cm, in acetone olive oil solution with 3.3% 2,4-dinitrochlorobenzene (DNCB) (proportion 1 : 1) Smear specimen to abdominal incision, once a day, 50μl each time, for 4 consecutive days. From the 5th day, insert a silicone tube with a diameter of 1 mm into the mouse colon with the tip of the cannula 3 to 3.5 cm away from the anus, and put 0.2 to 0.4% DNCB ethanol solution into the enema (ratio 3: 2). , Once a day, 2μl/g each time, for 4 consecutive days. During the modeling process, the body weight and fecal characteristics of the mice were observed every day, and the disease activity index (DAI) was used to score the animal's condition. ① Body weight: Normally, it is 0 points, which means a decrease of 1% to 5% and 6%. 10%, 11%-15%, 15%, 1 point, 2 points, 3 points, 4 points.

　　(2) Stool viscosity: 0 points for normal, 2 points for looseness, 4 points for diarrhea. ③Stool bleeding: 0 points for normal, 2 points for advantage. On the 9th day after the enema, blood was drawn by depilation, the serum was separated and the serum NO and TNF-a levels were detected; the abdominal cavity was opened, the large intestine was separated, and the intestinal wall adhered along the mesentery. Perform a vertical cut and observe the overall shape change. Tissue specimens were taken out from severe lesions, fixed in 10% formaldehyde solution, routinely embedded in paraffin, HE stained, and the pathological changes of the colon were observed under a microscope. Overall morphological scoring criteria: no colonic adhesions (the colon is easily separated from other tissues), no ulcers, no inflammation 0 points, mild colonic adhesions, 1 point local congestion, severe colonic adhesions and 1 ulcer (1 point, 1 cm inflammation) Can earn 4 points. Microscopic pathological scoring criteria: normal colonic mucosa covered 0 grade, crypt defect 1/3 grade, crypt defect 2/3 grade 2, epithelial monolayer, mild inflammatory cell infiltration infiltrated layer 3 grade. Mucosal erosions are grade 4 ulcers, with obvious infiltration of inflammatory cells. Alternatively, adult male rats should be depilated with 10% Na2S solution on the nape of the neck, and then drop 2% DNCB acetone on the back once a day for 5 consecutive days, 5 drops each time. Subsequently, a nylon catheter was used to inject a 0.1% DNCB ethanol solution into the 8 cm colon cavity of the anus once a day at a rate of 0.25 ml/time/day for 4 consecutive days. After modeling, observe the overall condition of the model animals and the performance of gastrointestinal diseases every day. I sacrifice a few animals every week. Observe the appearance of the colonic mucosa and serosal surface with naked eyes, and remove and fix the lesion. Use 10% formaldehyde solution. Normal paraffin-embedded sections, HE staining, and optical microscopy to determine the shape, depth, type, degree of infiltration of inflammatory cell ulcers in each layer of the intestinal wall, changes in lymphoid follicular hyperplasia and the formation of fibrous tissue hyperplasia. Alternatively, in adult male guinea pigs, use a 2% DNCB ethanol solution to cut off about 1 square centimeter of hair on the back of the neck once a day, and drop it on the surface, 5 drops each time for 10-14 days. Subsequently, a silicone tube with a diameter of 3 mm was inserted into the intestine at a depth of 12 cm from the anus, 0.5 ml of 20% DNCB acetone solution was injected, and the animals were sacrificed 2 days later. The materials, observation and evaluation methods are the same as above.

　　(3) Model characteristics: Model mice began to have enema after 24 hours and lost significant weight after 3 days. On day 5, a 0.2% DNCB enema showed intestinal adhesions and flatulence. I observed it with a microscope. The glands are arranged chaotically and goblet cells are reduced. Diffuse inflammatory cell infiltration in the lamina propria, crypt abscess and ulcer is clearly visible. 0.4% of patients with DNCB have extensive adhesions in the colon, the proximal end of the intestinal cavity is dilated, a small amount of white exudate, the colonic mucosa is hyperemia, necrosis, and the formation of multiple ulcers. The lower crypts have obvious defects, disordered tissue structure, mucosal erosion, hemorrhage, necrosis, and large deep ulcers. .. Model rats have four consecutive enemas, lack of energy, discomfort, roundness, loose hair, reduced activity, reduced food consumption, sparse feces or mucus, and bloody stools. Within 12 days of the last edema, the colonic mucosa of model rats showed different degrees of hyperemia and edema, and its surface was covered with mucus, blood and necrotic tissue, and there were obvious ulcers; the mucous membranes surrounded the ulcers. Submucosal congestion and edema, severe congestion and edema. Or moderate neutrophil and eosinophil infiltration, and a small to moderate amount of large monocytes and lymphocyte infiltration; the bottom of the ulcer is mainly angiogenic granular tissue. Some have different degrees of fibrous tissue hyperplasia; the muscle layer is also infiltrated with a small to moderate amount of neutrophils and eosinophils, and occasionally large monocytes and lymphocytes infiltrate. Twelve to fifty days after the completion of the model, the colon of the model animal still showed infiltration and edema, but its degree gradually weakened with the passage of time; under a microscope, the colon mucosa and submucosa were slightly infiltrated and entered lymphocytes Eosinophils are examples: main inflammatory cell infiltration, occasionally neutrophils and large intestine infiltration; lymphocytes and eosinophils occasionally infiltrate into the muscle layer, fibrous tissue of the intestinal wall of some model animals, proliferation and Local mucosal atrophy, irregular shapes of crypts and intestinal epithelium regenerate. The histopathological changes of model guinea pigs are basically the same as that of model rats. The main symptoms are congestion, edema, thickening of the intestinal wall, a large number of neutrophils and other inflammatory cells that penetrate into the submucosa, crypt abscesses and ulcers. It can form and necrosis in severe cases.

　　(4) Comparative Medicine 2,4-Dinitrochlorobenzene (DNCB) is a small molecule chemical substance that can be used as a hapten that binds to tissue proteins to form a complete antigen, and stimulates the body to stimulate T cells. immune response. In this experiment, DNCB rubbed against the animal's epidermis and sensitized lymphocytes by binding to skin proteins. When sensitized lymphocytes come into contact with DNCB in the intestine again, the allergic reaction is delayed, similar to human ulcerative colitis. It manifests as diarrhea, blood in the stool, inflammation of the colonic mucosa, and even ulcers. The histopathological characteristics of the colonic mucosa of model animals are obviously related to the time after completing the model. Within 12 days after the completion of the model, colonic mucosa ulcers, surrounding tissues and muscle layers can show obvious inflammatory cell infiltration belonging to the ulcer phase; within 12-50 days, colonic mucosal inflammatory cell infiltration decreases, and some mucosa has obvious fibrous tissue. Intestinal wall hyperplasia is the recovery period of ulcers. Because the response to DNCB varies from species to species, the concentration, dose, and duration of action of DNCB vary greatly during the modeling process and should be repeatedly studied by the experimenter. This model is more suitable for studying the etiology of ulcerative colitis, the types and methods of inflammatory cells involved in the reaction. Although this model is not characterized by long cycles and recurrent lesions, it is one of the most widely used animal models of ulcerative colitis, as a typical animal model of delayed allergy in the gastrointestinal tract.