小干扰RNA对肝星状细胞基质金属蛋白酶组织抑制因子-1表达的抑制作用  

Suppression of Tissue Inhibitor of Metalloproteinase-1 on Gene Expression by Small Interfering RNA in Rat Hepatic Stellate Cell

作　　者：丛敏[1] 王萍[1] 刘天会[1] 徐雍[1] 卢炎[1] 唐淑珍[1] 刘晓明[1] 王宝恩[1] 贾继东[1] 尤红[1] 

机构地区：[1]首都医科大学附属北京友谊医院肝病中心

出　　处：《首都医科大学学报》2006年第6期767-770,共4页Journal of Capital Medical University

基　　金：国家自然科学基金(30500425);北京市自然科学基金(7053066);北京市科技新星计划(2004B32);北京市"肝脏保护与再生调节"重点实验室资助项目

摘　　要：目的观察化学合成的小干扰RNA（siRNA）转染大鼠肝星状细胞（HSC）-T6后不同时间点，对基质金属蛋白酶组织抑制因子-1（TIMP-1）的抑制作用，同时筛选高效的siRNA片断。方法对大鼠TIMP-1 mRNA nt161～181、nt190～208、nt208—226、nt226～244及nt445～463化学合成的5对siRNA和1对荧光标记的非特异siRNA（与TIMP-1 mRNA无同源性的FITC标记的21nt siRNA），在阳离子脂质体的介导下将不同浓度的非特异siRNA转染至大鼠HSC-T6后，用流式细胞仪确定最佳转染浓度。提取转染50nmol／L siRNAs后24h，48h和72h细胞蛋白，用Western blot检测TIMP-1蛋白质表达，筛选出抑制效率最高的siRNA，同时确定siRNA转染细胞后的最佳作用时间。结果50nmoL／L siRNAs对HSC-T6有较高的转染效率；5对siRNA中的3对在转染后48h有较强的抑制HSC-T6细胞TIMP-1基因表达的作用，其中抑制作用最强的1对siRNA对TIMP-1表达的抑制作用与对照组相比可增强80％以上，而在转染后72h，其抑制作用基本消失。结论化学合成的siRNA在短时期内可有效地抑制TIMP-1基因的表达，筛选到的高效阻抑TIMP-1表达的siRNA可构建于病毒载体，以实现长期抑制TIMP-1表达的功效。Objective To investigate the effects of RNA interference targeting TIMP-1 gene on rat hepatic stellate cell（HSC）-T6 cells in vitro and screen highly efficient small interfering RNA（siRNA）. Methods Five pair of 21 nucleotide siRNAs targeting rat TIMP- 1 at nt161 - 181, nt 190 - 208, nt 208 - 226, nt 226 - 244, nt 445 - 463 and one pair of fluorescence labeled unspecific siRNA were synthesized. After transfecting different concentrations of unspecific siRNA to select the optimum transfection concentration, 50 nmol/L siRNAs were transfected into rat HSC-T6 cells respectively. The protein of the infected cells at 24 h, 48 h and 72 h was collected and detected by Western blot to confirm the suppression effects of diverse siRNAs at different time point. Results It was demonstrated by fluorescence actived cell sorter that 50 nmol/L siRNA had the optimum trasfection efficacy, and 3 of the 5 siRNA oligomers had the significant effects on suppressing TIMP-1 expression by Western-blot within 48 hours after transfecting into rat HSC-T6 cells. The expression level of TIMP-1 gene in HSC-T6 cells infected by one pair of siRNA after 48 h decreased significantly （80%） compared with control cells, and the effects of suppression disappeared at 72 h after transfection. Conclusion RNA interference can exert an suppression of TIMP-1 gene in rat HSC. One pale of siRNA, which can highly effectively inhibit expression of TIMP-1 gene was screened successfully, and it will be reconstructed into virus vectors to suppress the specific gene expression for a long time by chromosomal integration.

关 键 词：金属蛋白酶组织抑制因子-1 小干扰RNA 肝星状细胞 

分 类 号：TQ937[化学工程]