动物模型,溃疡性结肠炎 z-bo 2020-08-07 1119

　　(1) Reproduction method BALB/c mice, 7-8 weeks old, weighing 25-30g, shaved the abdominal skin (2cm×2cm), and smeared with 0.2ml of 3% oxazolone (dissolved in 100% ethanol) Skin, repeat once every other day, 5 days later, insert a 2mm diameter silicone tube from the anus into the intestine about 4cm deep, and inject 0.15ml of 1% oxazolone (dissolved in 50% ethanol). After enema, the mice's body weight, stool characteristics and blood in the stool were observed daily, and the disease activity index (DAI) score was performed. The animals were sacrificed at 24h, 3, 7, 14, 21 days after the enema, the colon was opened immediately, the intestinal cavity was cut along the mesenteric margin, washed, and some tissues of the colon where the lesions were obvious were taken for morphological examination, and some tissues were treated as marrow Peroxidase (MPO) activity and cytokine TNF-a, IFN-7, IL-4 content determination. DAI scoring criteria: ①Body weight, stool characteristics, occult blood or bloody stools are all normal: 0 points. ② Weight loss of 1% to 5%, stool characteristics, occult blood or bloody stools are normal: 1 point. ③ Weight loss of 5% to 10%, mushy stool, positive occult blood: 2 points. ④ 10% to 15% weight loss, semi-sparse stool, positive occult blood: 3 points. ⑤ Weight loss is greater than 15%, stool watery, bloody stool: 4 points. The degree of histological damage was scored by the sum of the scores of items such as epithelial injury, ulcer formation, and inflammatory cell infiltration. ① Epithelial injury and ulcer formation (ulcer depth): none: 0 points; erosion (submucosal layer): 1 point; ulcer (muscular layer): 2 points; ulcer (serosa layer): 3 points. ② Edema: none: 0 points; mild: 1 point; moderate: 2 points; severe: 3 points. ③ Lymphatic, mononuclear and plasma cell infiltration (infiltration depth): none: 0 points; mild (submucosal layer): 1 point; moderate (muscular layer): 2 points; severe (serosa layer): 3 points. ④ Neutrophil infiltration (infiltration depth): none: 0 points; mild (submucosal layer): 1 point; moderate (muscular layer): 2 points; severe (serosal layer): 3 points. ⑤ Eosinophil infiltration (depth of infiltration): none: 0 points; mild (submucosa): 1 point; moderate (muscular layer); 2 points; severe (serosal layer): 3 points. MPO activity determination: cut out part of the diseased colon tissue and weigh it, add 1ml of 0.2% cetyltrimethylammonium bromide (HTAB) per 200mg, homogenize and freeze-thaw for 3 times, centrifuge at 1083g for 10min, take the supernatant 0.1 ml, with o-linked [di]anisidine as the substrate, the average value of the absorbance change in 5 minutes was measured with a UV-200 ultraviolet spectrophotometer at a wavelength of 655mm, and the change in absorbance value per minute was 1.0 unit of enzyme activity. Cytokines TNF-a, IFN-γ and IL-4 content determination: cut out part of the diseased colon tissue and weigh it, add 1ml PBS (pH 6.0, containing aprotinin, leupeptin and pepstatin A each 50mg) 1ug) homogenize, centrifuge at 6500g at 4°C for 15min, take 0.1ml of the supernatant, and use the corresponding kit to determine the content of cytokines TNF-a, IFN-γ and IL-4 by ELISA.

　　(2) Model characteristics: Two days after the mice were applied with oxazolone for two times, the skin of the applied area may have redness and swelling, and some of the skin may be ulcerated. Weight loss and diarrhea occurred 24 hours after the enema. On the 3rd to 4th day The diarrhea reached a peak, some animals had gross bloody stools, the diarrhea lasted for about 1 week and then gradually turned into soft stools, and the stool characteristics returned to normal after 2 weeks. In addition, 24 hours after enema, congestion and edema of the distal colonic mucosa appeared, and the lesions were distributed continuously. Microscopically, they showed loss of epithelial cells, erosion and superficial ulcer formation, decreased goblet cells, decreased gland density, and inflammation confined to mucosa and mucosa In the lower layer, the lamina propria of the mucosa can be seen with a variety of inflammatory cell infiltrations. In the early stage, neutrophil infiltration is dominant. After 1 week, lymphocytes, monocytes and plasma cells are infiltrated, with a few neutrophils and eosinophils. ; Colon inflammation can last for about 2 weeks. At the same time, at 24h, 3d and 7d after enema, the MPO activity and IL-4 content of the diseased colon tissue were significantly increased, while the TNF-a and IFN-γ content were basically normal.

　　(3) Comparative medicine Oxazolone is a kind of hapten. When it is combined with skin protein, it can form a complete antigen. Through the strong antigen presentation of skin dendritic cells, namely Langerhans cells, the main tissue is compatible The antigens on the MHC class II molecules are presented to CD4+ T cells. These T cells proliferate to form T cell clones specific to the hapten-modified self-peptide. When the body is exposed to oxazolone again, the modified The self-peptide of T cells presents the MHC class II molecules on the dendritic cells again, and memory T cells release cytokines to produce an immune response to these antigens, and induce T cell-mediated delayed-type allergic reactions, leading to intestinal inflammation. The use of oxazolone to induce animal colitis is a newly established model of ulcerative colitis similar to humans. There are two methods for making molds, one is a one-time enema; the other is to sensitize first and then enema. Due to the short duration of colon inflammation caused by the former method (3 to 4 days) and the higher mortality rate in mice, it is more common to use the latter method to make models. This model replication method showed that after applying oxazolone, the local skin of the animal appeared red and swollen. After 5 days, the small dose of oxazolone was administered again by enema, and intestinal inflammation soon appeared. In this method, it is the gastrointestinal mucosa that is exposed to oxazolone again, and the mucosal inflammation caused by this is more severe and lasting longer. Due to the simple modeling method, good reproducibility, and the histological characteristics and inflammation distribution of this model are similar to similar human diseases, it also replicates the chronic and recurrent characteristic inflammation pathogenesis well, which is the etiology and pathogenesis of ulcerative colitis The research provides a good method, which can also be used for the evaluation of drug activity and efficacy.