反义及小干扰RNA对大鼠肝星状细胞金属蛋白酶组织抑制因子-1表达的抑制作用  

Comparison between the suppression of tissue inhibitor of metalloproteinase-1 gene expression by recombinant adenoassociated virus carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 in rat hepatic stellate cells

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,北京100050

出　　处：《中华肝脏病杂志》2006年第10期742-747,共6页Chinese Journal of Hepatology

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);北京地区高等学校"肝脏保护与再生调节"重点实验室资助

摘　　要：目的观察以腺相关病毒(AAV)为载体,含有针对大鼠金属蛋白酶组织抑制因子(TIMP)-1的反义RNA及小干扰RNA(siRNA)的重组AAV(rAAV/ANTI-TIMP-1/neo及rAAV/siRNA-TIMP-1/neo)感染大鼠肝星状细胞系HSC-T6后,TIMP-1表达的受抑制情况。方法经聚合酶链反应(PCR)扩增及酶切后连接,将针对TIMP-1的反义RNA片段及siRNA片段分别构建于AAV载体中并测序鉴定,将其包装成重组病毒后感染大鼠肝星状细胞系HSC- T6,同时设空白对照组。经G418以400μg/ml的浓度筛选30d后,分别应用荧光定量PCR技术及Western blot检测重组病毒感染组及空白对照组HSC-T6 TIMP-1的基因转录及蛋白质表达水平。结果经PCR、酶切及序列测定证实,两种重组AAV载体质粒(pd16-95/ANTI-TIMP-1/neo和pd16-95/siRNA-TIMP—1/neo)克隆成功。将重组质粒包装成病毒感染HSC-T6细胞30 d后,通过荧光定量PCR技术及Western blot分析显示,重组病毒rAAV/siRNA-TIMP- 1/neo组与对照组细胞相比,HSC-T6中的TIMP-1基因的转录被抑制(P＜0．01),且表达水平与对照组相比约下降60%；而rAAV/ANTI-TIMP-1/neo组及空载体组与对照组相比TIMP-1基因的转录及表达水平差异无统计学意义(P＞0．05)。结论通过AAV载体技术重组病毒rAAV/siRNA-TIMP-1/neo可有效地抑制TIMP-1基因的表达,而针对TIMP-1基因全长的重组病毒rAAV/ANTI-TIMP-1/neo对体外培养的HSC-T6细胞TIMP-1基因的转录与表达无明显抑制作用。Objectives Elevated tissue inhibitor of metalloproteinase-1 （TIMP- 1） expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses （AAV） carrying antisense RNA and small interfering RNA （siRNA） of TIMP- 1 （rAAV/ANTI-TIMP- l/neo and rAAV/siRNA-TIMP- 1/ neo）, and then to compare the suppression of TIMP- 1 gene expression on rat hepatic stellate cell （HSC）-T6 cells infected by these two types of viruses in vitro. Methods Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector （pd16-95/neo） and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP- 1/neo and rAAV/siRNA-TIMP-1/rico. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells. Results The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pd16-95/ANTI-TIMP-1/neo and pd16-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP- 1/neo and rAAV/siRNA-TIMP- 1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TLMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells （P 〈 0.01）, and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly （60%）, while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANT1- TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells （P 〉 0.05）. Conclusions RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integr

关 键 词：金属蛋白酶组织抑制因子-1 RNA 反义 小干扰RNA 腺相关病毒 肝星状细胞 

分 类 号：R575.2[医药卫生—消化系统]