动物模型,溃疡性结肠炎 z-bo 2020-08-07 535

　　(1) Replication method Adult male BALB/c mice, cut their abdominal hair (without damage to the skin) about 2.0cm×1.5cm, and use 3.3% 2,4-dinitrochlorobenzene (DNCB) acetone olive oil solution (ratio 1: 1) Abdomen smear, 1 time/d, 50μl/time, for 4 consecutive days. From the 5th day, insert a silicone tube with a diameter of 1mm into the mouse colon, with the tip of the cannula 3～3.5cm away from the anus, and enema with 0.2～0.4% DNCB ethanol water solution (ratio 3:2), once a day, each time 2μl/g, continuous 4d. In the process of modeling, observe the weight and stool characteristics of the mice daily, and use the disease activity index (DAI) to score the animal status: ①Weight: Normal is 0 points, which is 1% to 5%, 6% lower than normal -10%, 11%-15%, >15%, respectively, 1 point, 2 points, 3 points, 4 points. ②Stool viscosity: 0 points for normal, 2 points for looseness, and 4 points for diarrhea. ③Stool bleeding: 0 points for normal and 2 points for dominant. On the 9th day after the enema, blood was taken by decapitation, the serum was separated, and the levels of NO and TNF-a in the serum were detected; the abdominal cavity was opened, the large intestine was separated, the intestinal wall was cut longitudinally along the mesenteric attachment, and the gross morphological changes were observed. Tissue specimens were taken from severe lesions, fixed in 10% formaldehyde solution, embedded in conventional paraffin, HE stained, and the pathological changes of the colon were observed under microscope. Gross morphological scoring criteria: 0 points for no colon adhesion (the colon is easily peeled off from other tissues), no ulcers, no inflammation, 1 point for mild colonic adhesions and local congestion, and 1 point for colonic adhesions and 1 ulcer (<1cm) 2 points, 3 points for more than 1 ulcer (<1cm) 4="" with="" points="" for="" multiple="" ulcers="">1cm) with inflammation. Microscopic pathology scoring criteria: normal colonic mucosa is grade 0, crypt defect 1/3 is grade 1, crypt defect 2/3 is grade 2, and lamina propria covered with a monolayer of epithelium with mild inflammatory cell infiltration is grade 3. Mucosal erosions and ulcers with significant inflammatory cell infiltration are grade 4. Or adult male rats, after depilating the back of the neck with 10% Na2S solution, use 2% DNCB acetone drops on the back, 1 time/d, 5 drops/time, for 14 consecutive days. Subsequently, a nylon catheter was used to inject 0.1% DNCB ethanol solution into the 8cm colonic lumen of the anus, once/d, 0.25ml/time, for 4 consecutive days. After modeling, observe the overall appearance of the model animals and the performance of digestive tract diseases every day. Several animals were sacrificed every week. The appearance of the colonic mucosa and serosal surface was observed with naked eyes, and the lesions were cut and fixed with 10% formaldehyde solution. Routine paraffin-embedded sections, HE staining, light microscope examination of ulcer shape, depth, types of inflammatory cells in the intestinal wall and degree of infiltration, as well as changes in lymphoid follicular hyperplasia and fibrous tissue hyperplasia. Or adult male guinea pigs, cut off about 1 square centimeter of hair on the back of the neck, and drip on the surface with 2% DNCB ethanol solution, 1 time/d, 5 drops/time, for 10-14 days. Subsequently, a silicone tube with a diameter of 3 mm was taken and inserted into the intestine 12 cm deep through the anus, and 0.5 ml of 20% DNCB acetone solution was injected, and sacrificed 2 days later. The materials, observation and evaluation methods are the same as above.

　　(2) Model features Model mice began to develop diarrhea after enema 24 hours, and their body weight decreased significantly after 3 days. On the 5th day, 0.2% DNCB enema had intestinal adhesion and flatulence. Under the microscope, it was seen that the glands were arranged disorderly, goblet cells decreased, and the mucous membrane was inherent. Diffuse inflammatory cell infiltration, crypt abscesses and ulcers are clearly visible, 0.4% DNCB patients have extensive colonic adhesions, proximal intestinal cavity dilation, a small amount of white exudate, colonic mucosa hyperemia, necrosis, multiple ulcer formation, hidden under the microscope The fossa defect is obvious, the tissue structure is disordered, the mucosa is erosion, hemorrhage, necrosis and large area deep ulcer. Model rats had 4 consecutive enemas, lack of energy, fatigue, curled up, loose fur, reduced activity, decreased food consumption, loose stools or mucus and blood in the stool. Within 12 days after the last enema, the colonic mucosa of the model rats showed varying degrees of hyperemia and edema, and the surface was covered with mucus, blood and necrotic tissue, and there were obvious ulcer formation; the mucosa and submucosa surrounding the ulcer were congested and edema, and there was a lot of Or a moderate amount of neutrophils and eosinophils, and a small to moderate amount of large monocytes and lymphocytes infiltration; the bottom of the ulcer is mostly vascularized granulation tissue, and some have different degrees of fibrous tissue proliferation; also in the muscle layer A small to moderate amount of neutrophils and eosinophils infiltrate, and occasionally large monocytes and lymphocytes infiltrate. 12-50 days after the completion of the model, the model animal’s colon still showed congestion and edema, but the degree gradually reduced with time; under the microscope, the colon mucosa and submucosa tissues were slightly congested, with lymphocytes and eosinophils as Main inflammatory cell infiltration, occasional infiltration of neutrophils and large monocytes; occasional infiltration of lymphocytes and eosinophils in the muscle layer, fibrous tissue proliferation in the intestinal wall of some model animals, and local mucosal atrophy, Irregular shape of crypts and intestinal epithelial regeneration. The histopathological changes of the model guinea pig are basically the same as those of the model rat. The main manifestations are intestinal wall congestion, edema, thickening, infiltration of a large number of neutrophils and other inflammatory cells in the submucosa, formation of crypt abscesses and ulcers, and necrosis in severe cases.

　　(3) Comparative Medicine 2,4 Dinitrochlorobenzene (DNCB) is a small molecule chemical substance, which can be used as a hapten and tissue protein to combine to form a complete antigen, which stimulates the body to produce a cellular immune response mediated by T cells. In this experiment, DNCB was rubbed on the epidermis of animals, and the lymphocytes were sensitized by binding to skin proteins. When the sensitized lymphocytes contact DNCB again in the intestines, delayed allergies will occur, causing similar human ulcerative colitis The pathological changes, manifested as diarrhea, blood in the stool, inflammation of the colonic mucosa and even ulcers. The histopathological characteristics of the colon mucosa of model animals are obviously related to the time after the completion of the model. Within 12 days after the completion of the model, the colonic mucosa ulcers, the surrounding tissues and muscle layer can see significant inflammatory cell infiltration, which belongs to the ulcer stage; at 12-50 days, the colonic mucosal inflammatory cell infiltration is reduced, and part of the intestinal wall has obvious fibrous tissue Hyperplasia is the recovery period of ulcers. Because different species have different responses to DNCB, the concentration, dose, and duration of action of DNCB vary greatly during the model making process, which requires experimenters to explore repeatedly. This model is more suitable for the study of the pathogenesis of ulcerative colitis and the types and ways of inflammatory cells participating in the reaction. Although this model has a long period and does not have the characteristics of easy recurrence of lesions, it is still one of the most widely used animal models of ulcerative colitis as an animal model for a typical delayed-type gastrointestinal allergic reaction.