失血性休克对大鼠肠黏膜屏障Occludin表达的影响

Influence of hemorrhagic shock on tight junction protein Occludin in intestinal mucosa of rats

目的探索失血性休克对肠黏膜细胞紧密连接的作用以及对Occludin表达的影响。方法sD大鼠24只，按随机数字表法分为假手术组、失血性休克0．5h组、2h组、4h组，每组6只。采用Wiggers改良法制备失血性休克大鼠模型。腹主动脉取血，活性比色法测定血浆二胺氧化酶（diamine oxidase，DAO）活性；分光光度计法测定血浆D-乳酸（D—Lactate，D—LA）含量；比浊法测定血浆内毒素含量；肠系膜淋巴结和肝组织细菌培养检测细菌移位情况；光镜观察大鼠肠黏膜病理变化；电镜观察肠上皮细胞超微结构；RT—PCR测定小肠黏膜Occludin的mRNA表达；Westernblot与免疫组化法测定Occludin蛋白表达。结果失血性休克0．5h组DAO、D—LA及内毒素含量分别为（72．68±10．24）mg／ml、（9．28±0．53）μg／ml、（41．25±3．68）pg／L，较假手术组明显升高（P〈0．05）。2h组肝组织和肠系膜淋巴结均培养出细菌，4h组菌落量明显增高（P〈0．01）。小肠上皮的形态及超微结构在休克0．5h组即发生破坏，随着失血时间延长，破坏越严重。失血性休克0．5h组肠黏膜OccludinmRNA降低（P〈0．01）；2h组降低更明显（P〈0．05）。失血性休克0．5h组Occludin蛋白定位表达强度呈弱阳性；2h组Occludin蛋白量较0．5h组明显降低（P〈0．01），定位表达亦呈阴性。结论Occludin分子在大鼠肠黏膜上皮细胞中的表达随失血性休克的时间延长下降，推测可能是导致细胞紧密连接损伤的重要基础。

Objective To investigate the effect of hemorrhagic shock on intestinal epithelial tight junction and expression of protein Occludin. Methods Twenty-four SD rats were allocated to sham operation group, hemorrhagic shock 0.5- hour, 2-hour, and 4-hour groups according to random number table, with 6 rats per group. A model of hemorrhagic shock in the rat was induced by the improved Wiggers method. Blood samples were drawn from abdominal aorta. Activity of plasma diamine oxidase （DAO） was determined by active colorimetric method; content of plasma D-lactic acid （D-LA） by spectrophotometer method ; plasma endotoxin levels by turbidimetry ; bacterial translocation by bacterial culture of mesenteric lymph nodes and liver tissues; pathological changes of intestinal mucosa by light microscope; uhrastructure of intestinal epithelial cells by electron microscopy; Occludin mRNA expression in small intestinal mucosa by RT-PCR; Occludin expression by Western blot and immunohistochemical method. Results Levels of DAO [ （72.68±10.24） mg/ml ], D-LA [ （9.28 ±0.53 ） pμg/ml ]and endotoxin [ （41.25±3.68 ） pg/L]in hemorrhagic shock 0.5- hour group increased markedly as compared with those in sham operation group （P 〈 0.05）. Bacteria were cultivated from the liver tissues and mesenteric lymph nodes in hemorrhagic shock 2-hour group and much more colonies of bacteria were observed in hemorrhagic shock 4-hour group （P 〈 0.01 ）. Morphology and uhrastructure of the small intestinal epithelium manifested immediate damage in hemorrhagic shock 0.5- hour group and much worse detriment was observed with a prolonged bleeding time. Occludin mRNA expression in small intestinal mucosa decreased in hemorrhagic shock 0.5- hour group, followed by a further reduction in hemorrhagic shock 2-hour group （ P 〈O. 05 ）. Localized expression of Occludin in hemorrhagic shock O. 5- hour group was weakly positive. Occludin content in hemorrhagic shock 2- hour group showed significant reduction when compared with that in hemorrhagic shock O. 5- hour group （P 〈0. O1 ） and its localized express was negative as well. Conclusion Occludin expression in intestinal epithelial cells decreases obviously with a prolonged bleeding time, which is presumed to be an important basis for disruption of cellular tight junction.