去乙酰化酶Sirt3沉默对缺氧条件下肠上皮Caco-2细胞缺氧诱导因子-1α表达的影响及其对肠上皮屏障功能的作用  

Effects of silencing deacetylase Sift3 on hypoxia inducible factor-1α and epithelial barrier function of Caco-2 cells under hypoxia

作　　者：逯顶松 杨松巍[1] 陈国庆[1] 刘勇[1] 马远航[1] 杨桦[1] 

机构地区：[1]第三军医大学新桥医院普通外科,重庆400037

出　　处：《中华实验外科杂志》2014年第2期282-285,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金重大国际合作项目（81020108023）;国家自然科学基金资助项目（81272078）;重庆市科委国际合作重点项目（201110008）

摘　　要：目的 采用RNA干扰（RNAi）技术,使去乙酰化酶Sirt3基因表达下调或缺失,观察其下调或缺失后对缺氧诱导因子-1α（HIF-1α）表达的影响及其对肠屏障功能的作用.方法 体外培养Caco-2细胞,分别检测跨上皮电阻（TER）,常氧和低氧（1％O2）条件下Sirt3-小干扰RNA（siRNA）转染前后HIF-1α和紧密连接蛋白：ZO-1、Occludin、Claudin-1的mRNA与蛋白表达.结果 常氧与低氧条件下,Sirt3-siRNA转染后Sirt3的蛋白表达分别下降52％、35％;HIF-1α的蛋白表达（0.72±0.01、0.93 ±0.01）比转染前（0.38 ±0.01、0.81±0.03）明显升高（P＜0.01）;低氧条件下,Sirt3-siRNA转染后Occludin、Claudin-1、ZO-1的蛋白（0.71 ±0.03、0.69±0.01、0.72±0.02）和mRNA （0.80±0.01、0.84±0.01、0.88 0.03）表达比转染前明显降低（P＜0.01）;低氧条件下,经Sirt3-siRNA转染后TER下降30.8％.结论 缺氧条件下,Sirt3可以调控HIF-1α的表达.沉默Sirt3后紧密连接蛋白表达降低,从而影响肠屏障功能.Objective To investigate the effects of silencing Sirt3 on hypoxia-inducible factor-1α（HIF-1α） and tight junctions （TJs） of Caco-2 cells.Methods The Caco-2 cells were cultured in vitro,and then divided into 4 groups：the normoxia group （Nx）,the normoxia plus Sirt3-siRNA group,the hypoxia group （Hx,1% O2 for 6 h）,and the hypoxia plus Sirt3-siRNA group.Transepithelial electrical resistance （TER） was determined.The protein expression of Sirt3 and HIF-1α was detected by using Western blotting.Occludin,Claudin-1 and zonulaoccludens-1 （ZO-1） expression was examined by using reverse transcription polymerase chain reaction （RT-PCR） and Western blotting.Results Sirt3 protein expression was significantly reduced （52%,35%） after transfection of Sirt3-siRNA.The expression levels of HIF-1α protein （0.93 ± 0.01） was increased in the hypoxia plus Sirt3-siRNA group as compared with those under hypoxia for 6 h and normoxia.The expression of TJs mRNA （ZO-1：0.88 ±0.03,Occludin：0.80±0.01,and Claudin-1：0.84±0.01） （P〈0.01）,andTJS protein （ZO-1：0.72 ±0.02,Occludin：0.71 ±0.03,Claudin-1：0.69 ±0.01） （P 〈0.01） were decreased after Sirt3-siRNA transfection as compared with that before transfection.The TER value in the hypoxia plus Sirt3-siRNA group was decreased by 30.8% as compared with hypoxia for 6 h.Conclusion Sirt3 could regulate the expression of HIF-1α and TJs under hypoxia,and then influence the epithelial barrier function.

关 键 词：上皮屏障功能 RNA干扰 去乙酰化酶Sirt3 缺氧诱导因子-1 紧密连接蛋白 

分 类 号：R363[医药卫生—病理学]