中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
2期
282-285
,共4页
逯顶松%杨松巍%陈国庆%刘勇%马远航%杨桦
逯頂鬆%楊鬆巍%陳國慶%劉勇%馬遠航%楊樺
록정송%양송외%진국경%류용%마원항%양화
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
上皮屏障功能%RNA榦擾%去乙酰化酶Sirt3%缺氧誘導因子-1%緊密連接蛋白
상피병장공능%RNA간우%거을선화매Sirt3%결양유도인자-1%긴밀련접단백
Epithelial barrier function%RNA interference%Deacetylase Sirt3%Hypoxia inducible transcription factor 1%Tight junctions
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.
目的 採用RNA榦擾(RNAi)技術,使去乙酰化酶Sirt3基因錶達下調或缺失,觀察其下調或缺失後對缺氧誘導因子-1α(HIF-1α)錶達的影響及其對腸屏障功能的作用.方法 體外培養Caco-2細胞,分彆檢測跨上皮電阻(TER),常氧和低氧(1％O2)條件下Sirt3-小榦擾RNA(siRNA)轉染前後HIF-1α和緊密連接蛋白:ZO-1、Occludin、Claudin-1的mRNA與蛋白錶達.結果 常氧與低氧條件下,Sirt3-siRNA轉染後Sirt3的蛋白錶達分彆下降52％、35％;HIF-1α的蛋白錶達(0.72±0.01、0.93 ±0.01)比轉染前(0.38 ±0.01、0.81±0.03)明顯升高(P＜0.01);低氧條件下,Sirt3-siRNA轉染後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)錶達比轉染前明顯降低(P＜0.01);低氧條件下,經Sirt3-siRNA轉染後TER下降30.8％.結論 缺氧條件下,Sirt3可以調控HIF-1α的錶達.沉默Sirt3後緊密連接蛋白錶達降低,從而影響腸屏障功能.
목적 채용RNA간우(RNAi)기술,사거을선화매Sirt3기인표체하조혹결실,관찰기하조혹결실후대결양유도인자-1α(HIF-1α)표체적영향급기대장병장공능적작용.방법 체외배양Caco-2세포,분별검측과상피전조(TER),상양화저양(1％O2)조건하Sirt3-소간우RNA(siRNA)전염전후HIF-1α화긴밀련접단백:ZO-1、Occludin、Claudin-1적mRNA여단백표체.결과 상양여저양조건하,Sirt3-siRNA전염후Sirt3적단백표체분별하강52％、35％;HIF-1α적단백표체(0.72±0.01、0.93 ±0.01)비전염전(0.38 ±0.01、0.81±0.03)명현승고(P＜0.01);저양조건하,Sirt3-siRNA전염후Occludin、Claudin-1、ZO-1적단백(0.71 ±0.03、0.69±0.01、0.72±0.02)화mRNA (0.80±0.01、0.84±0.01、0.88 0.03)표체비전염전명현강저(P＜0.01);저양조건하,경Sirt3-siRNA전염후TER하강30.8％.결론 결양조건하,Sirt3가이조공HIF-1α적표체.침묵Sirt3후긴밀련접단백표체강저,종이영향장병장공능.
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.
