小肠移植排斥反应对肠道紧密连接蛋白表达的影响

Expression of tight junction proteins in the graft after small intestine transplantation

目的 观察大鼠小肠移植排斥反应对肠道紧密连接蛋白表达的影响.方法 建立大鼠异位小肠移植模型,分为3组：假手术组、无排斥反应组和排斥反应组.通过大鼠活动状态及病理评分确定排斥反应程度.采用酶联免疫吸附试验（ELASA）法检测血浆中二胺氧化酶（DAO）、D-乳酸（D-LA）、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-2的水平.透射电镜观察肠道紧密连接的超微结构改变.应用实时定量聚合酶链反应（Real-time PCR）、Westem blot法检测紧密连接蛋白Occludin和ZO-1 mRNA和蛋白表达水平的变化.结果 排斥反应组随时间延长,病理评分增加[3 d：（32.67±2.34）分;5 d：（43.50 ±±2.07）分;7 d：（69.50±2.43）分],肠黏膜屏障功能损伤加重[DAO：3 d：（35.84±5.60）U/ml;5 d：（46.50±O.80） U/ml;7 d：（54.01±1.70） U/ml],TNF-α和IL-2大量释放,肠道紧密连接损伤严重,紧密连接蛋白Occludin和ZO-1 mRNA和蛋白表达水平降低.线性相关分析发现,病理评分、TNF-α、IL-2与紧密连接蛋白Occludin和ZO-1的表达呈负相关（P＜0.01）.结论 大鼠小肠移植排斥反应可使肠道功能受损、紧密连接断裂、紧密连接蛋白Occludin和ZO-1表达水平降低,且其蛋白表达水平与排斥反应程度呈负相关.

Objective To explore the expression of tight junction proteins after small intestine transplantation in rats.Methods The hetetotopic small intestine transplantation models in rats were divided into sham operation,non-rejection and rejection groups.The rejection reaction was confirmed by active situation and pathological analysis.The cytokines,diamine oxidase,D-lactic acid,tumor necrosis factor （TNF）-α and interleukin （IL）-2 were detected by using enzyme linked immunosorbent assay （ELISA）.Ultrastructural change of tight junction was observed under the transmission electron microscopy.Furthermore,the expression levels of Occludin and ZO-1 were detected by using real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting.Results In the rejection group,as time went on,pathological score was gradually increased （3 d：32.67 ± 2.34,5 d：43.50 ± 2.07,and 7 d：69.50 ± 2.43）,and the injury of intestinal barrier was aggravated [DAO at 3 d：（35.84 ± 5.60） U/ml,at 5 d：（46.50 ±0.80） U/ml,and at 7 d：（54.01 ± 1.70） U/ml],especially with the mass release of TNF-α and IL-2.Simultaneously,the expression levels of Occludin and ZO-1 mRNA and proteins were decreased.There was a negative correlation between pathological score,TNF-α and IL-2 with the expression of Occludin and ZO-1.Conclusion In the rat hetetotopic small intestine transplantation models,intestinal mucosal barrier and tight junction are impaired by the rejection reaction,and the expression of Occludin and ZO-1 is decreased along with the progression of rejection reaction in the intestinal tissues.