核糖体S6激酶对肝星状细胞内胶原表达的调控  

Regulatory effect of ribosomal S6 kinase on collagen type Ⅰ expression in rat hepatic stellate cells

作　　者：杨妙芳[1] 许小兵[1] 吴文娟[1] 张晓华[1] 朱人敏[1] 

机构地区：[1]南京军区南京总医院消化内科,江苏南京210002

出　　处：《第四军医大学学报》2008年第19期1753-1756,共4页Journal of the Fourth Military Medical University

基　　金：南京军区南京总医院科研基金重点项目(2005029)

摘　　要：目的:探讨核糖体S6激酶(p90RSK)调节肝星状细胞(HSC)Ⅰ型胶原表达的作用.方法:将设计的p90RSK特异性siRNA克隆至真核表达载体pAV-U6+27中,构建p90RSK的RNA干扰真核表达载体pAV-RSKi.将构建的pAV-RSKi通过脂质体瞬时转染HSC-T6,分别采用实时定量聚合酶链式反应和Western Blot方法检测细胞的p90RSK和Ⅰ型胶原mRNA和蛋白表达.将p90RSK的真核表达质粒(pMT2-RSK1)和pAV-RSKi分别与Ⅰ型胶原启动子荧光素酶报告基因质粒(pGL3-COL1A1)共转染原代HSC,48h后检测细胞的荧光素酶活性.结果:构建的pAV-RSKi能够有效抑制HSC-T6中p90RSK mRNA和蛋白表达,与对照组相比较分别减少了(70.20±4.04)%和(89.10±5.26)%(P<0.01).HSC-T6中Ⅰ型胶原的mRNA和蛋白的表达,与对照组相比较分别减少(61.80±3.96)%和(98.50±7.01)%(P<0.01).各组转染pMT2-RSK1,pAV-RSKi和空载质粒的HSC中,Ⅰ型胶原启动子活性之间的差异无统计学意义(P>0.05).结论:p90RSK参与了HSC中胶原的表达调控,可能成为肝纤维治疗的新靶点.AIM： To investigate the regulatory effect of 90-kDa ribosomal S6 kinase（p90RSK） on the expression of collagen type Ⅰ in the hepatic stellate cells （HSCs）. METHODS： Specific siRNA of p90RSK was designed and cloned into the pAV-U6 ＋ 27 to construct the RNAi eukaryotic expression vectors of p90RSK （ pAV-RSKi ）, then the recombinant RNAi vectors were transiently transfected into HSC-T6 via Lipofectamine 2000. Real- time quantitative PCR and Western Blot were used to measure the p90RSK and collagen type Ⅰ mRNA and protein expressions in transfected HSC-T6. The p90RSK expression vectors or RNAi vectors were cotransfected into primary HSCs with the reporter vectors（ pGL3-COL1A1 ） containing collagen type Ⅰ promoter. The luciferase activity of transfected HSCs was determined after 48 h culture. RESULTS： The specific RNAi eukaryotic expression vectors targeting p90RSK were successfully produced, which reduced the expression of p90RSK mRNA and protein expression to （ 70.20 ± 4.04 ） % and （ 89.10 ± 5.26 ） % compared with control respectively（ P 〈 0.01 ）. Simultaneously, the expression of collagen type I was down-regulated [ （61.80 ± 3.96）% , （98.50 ± 7.01 ） % accordingly, P 〈 0.01 ） ] in HSC-T6 after RNAi treatment. There were no significant changes in collagen type Ⅰ promoter activity among HSCs transfected with p90RSK expression vector, RNAi vector and empty vector independently （ P 〉 0.05 ）. CONCLUSION： P90RSK is involved in the regulation of abnormal expression of collagen type Ⅰ in HSCs, which may be a new target for hepatic fibrosis.

关 键 词：核糖体蛋白质S6激酶类 肝/细胞学 星形细胞 胶原Ⅰ型 RNA干扰 

分 类 号：R675.2[医药卫生—外科学]