不同RNA干预技术对人乳头瘤病毒11型E7基因的沉默作用  

Silencing of HPV11ET gene expression by different RNA interference technology

作　　者：陈贤祯[1] 程浩[1] 汤晓燕[1] 张行[1] 朱可建[1] 叶俊[1] 林爱华[1] 岑建萍[1] 金纳[1] 

机构地区：[1]浙江大学医学院附属邵逸夫医院皮肤科,杭州310016

出　　处：《中华皮肤科杂志》2007年第5期267-270,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金(30371289)

摘　　要：目的探讨RNA干预技术的两种形式在体外对人乳头瘤病毒（HPV）11型E7基因的沉默作用。方法化学合成1对小片段干扰RNA（siRNA-11E7）、同时构建3个短发夹状小RNA（shRNA）表达质粒（pRNAT-11E71^#、2^#、3^#），分别转染稳定表达HPV11E7的C57BL／6小鼠黑素瘤细胞系B16细胞株，采用实时荧光定量PCR技术检测细胞转染前后靶基因mRNA的表达水平。结果siRNA-11E7转染细胞后6、12、24、48、72、96、120h对靶基因表达的抑制率分别为12.60％、31.41％、41.93％、42.93％、74．35％、32.71％、29．00％；最佳作用浓度为25nmol／L（抑制率70.06％），最低作用浓度可至3.13nmol／L（抑制率47.00％）。以0．4μg pRNAT-11E71^#、2^#、3^#及上述3个质粒等量混合转染细胞72h后对靶基因表达的抑制率分别达44．52％、59．26％、89.62％、54.09％，阴性质粒无干预作用。pRNAT-11E73^#作用6、12、24、48、72、96、120h对靶基因表达抑制率分别为0、17．50％、40．90％、42．40％、61．90％、51．50％、0％；最佳作用剂量0．2μg。结论siRNA及shRNA表达载体均可在体外安全高效地沉默HPV11E7基因的表达，其干预作用存在一定的量效性和时效性，且可能存在最佳作用浓度（剂量）和最佳作用时间。Objective To investigate the silencing effect of two kinds of RNA interference （ RNAi ） technology on the expression of HPV11 E7 gene in vitro. Methods A pair of small interference RNA （ siRNA ） targeting HPV11 E7 gene, named siRNA-11E7, was chemically synthesized, and three short hairpin RNA （ shRNA ） expression plasmids, i.e. pRNAT-11E7 1^#, 2^#, 3^#, were constructed. Then a murine melanoma cell line, B 16, which stably expressed HPV11 E7 gene, was transfected respectively with the siRNA- 11E7 and pRNAT-11E7 1^#, 2^#, 3^#, etc. The level of E7 mRNA expression was detected in these cells by real-time fluorescent quantitative PCR before and after the transfection. Results siRNA-11E7 could inhibit the E7 gene mRNA expression in B16 cells by 12.60%, 31.41%, 41.93%, 42.93%, 74.35%, 32.71% and 29.00% at 6 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h after the transfection, respectively. The optimal dose ofsiRNA was 25 nmol/L with an inhibition rate of 70.06%, and the lowest effective dose was 3.13 nmol/L with an inhibition rate of 47%. The 0.4 μg ofpRNAT-11E7 1^#, 2^#, 3^# and a mix of the 3 plasmids could decrease the target gene expression by 44.52%, 59.26%, 89.62%, 54.09%, respectively, while the negative plasmid had no inhibitory effect on the gene expression, pRNAT-11E7 3^# could decrease the E7 gene expression by 0, 17.50%, 40.90%, 42.40%, 61.90%, 51.50% and 0, at 6 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h after the transfection, respectively. The optimal dose ofpRNAT-11E7 was 0.2μg. Conclusions Both siRNA and shRNA expression plasmids could efficiently and safely silence-the HPV11E7 mRNA expression in a time- and dose-dependent manner, and the optimal action dose and time is likely to be identified.

关 键 词：基因沉默 RNA干预 乳头状瘤病毒 人 

分 类 号：R752.53[医药卫生—皮肤病学与性病学]