水通道蛋白3与紧密连接的相关性  

Lentiviral-mediated delivery of shRNA targeting aquaporin 3 alters tight junction protein expression and distribution in Caco-2 cells

作　　者：张文杰[1] 徐勇[1] 王斌[1] 徐皓[1] 

机构地区：[1]南京医科大学第一附属医院胃肠外科,江苏省南京市210029

出　　处：《世界华人消化杂志》2012年第21期1973-1977,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30901421;江苏省卫生厅开放课题基金资助项目;No.XK03200903~~

摘　　要：目的:探讨水通道蛋白3(aquaporin 3,AQP3)对肠黏膜上皮细胞间紧密连接(tight junction,TJ)的影响,并探讨其可能的作用机制.方法:应用Caco-2细胞系在体外构建肠黏膜上皮屏障,构建沉默AQP3的shRNA慢病毒载体,建立稳定转染细胞系.实验分为3组:空白对照组(BLANK)、阴性对照组(NC)、AQP3干扰组(AQP3 shRNA).Western blot验证TJ相关蛋白Occludin以及Claudin-1的表达情况;并且采用免疫细胞化学法观察TJ相关蛋白的分布和结构变化.结果:RT-PCR及Western blot结果显示在Caco-2细胞系中成功沉默AQP3的表达.干扰组与对照组相比下降约75%.Western blot结果显示AQP3干扰组TJ相关蛋白Occludin以及Claudin-1的表达明显降低.免疫细胞化学结果显示Caco-2细胞间Occludin以及Claudin-1主要表达在细胞膜和/或胞浆中,Occludin和Claudin-1细胞间棕褐色颗粒减少,结构变模糊.相邻Caco-2细胞间TJ结构遭到破坏.结论:靶向AQP3的shRNA技术可以引起TJ的结构变化和相关蛋白的表达分布的异常.AIM：To investigate the effect of lentiviral-mediated delivery of short hairpin RNA（shRNA） targeting aquaporin 3（AQP3） on the barrier function of intestinal tight junctions and to explore the possible mechanisms involved.METHODS：A lentiviral vector expressing shRNA targeting AQP3 was constructed and transfected into Caco-2 cells.The cells were divided into three groups：blank group,negative control group,and AQP3 shRNA group.Western blot and immunocytochemistry were used to detect the expression of occludin and claudin-1 proteins in transfected cells.RESULTS：RT-PCR and Western blot results showed that AQP3 expression was down-regulated in Caco-2 cells transfected with the lentiviral vector expressing shRNA targeting AQP3.Western blot analysis revealed that the expression levels of occludin and claudin-1 proteins were significantly decreased in Caco-2 cells transfected with the lentiviral vector expressing shRNA targeting AQP3.Immunocytochemistry showed that occludin and claudin-1 were mainly distributed in the cytoplasm and membrane.The structure of tight junctions was destroyed in cells transfected with the lentiviral vector expressing shRNA targeting AQP3.CONCLUSION：Lentiviral-mediated delivery of shRNA targeting AQP3 causes abnormal distribution of tight junction proteins and destruction of tight junctions in Caco-2 cells.

关 键 词：水通道蛋白3 CACO-2 紧密连接 

分 类 号：R363[医药卫生—病理学]