乙酰肝素酶基因siRNA表达载体的构建及其沉寂作用  

Construction of heparanase gene-targeted small interfering RNA and its gene silencing effect

作　　者：刘晓艳[1] 方红[1] 羊正纲[2] 张妤[1] 阮黎明[1] 蒋筱凌[1] 

机构地区：[1]浙江大学医学院附属第一医院皮肤科,浙江杭州310003 [2]浙江大学医学院附属第一医院传染病研究所,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2007年第6期581-587,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省教育厅资助项目(20051085)

摘　　要：目的:构建针对人乙酰肝素酶(HPSE)基因的小干扰RNA(siRNA)及其表达载体,转染细胞后观察其对HPSE基因的干扰作用。方法:设计HPSE靶向的发夹状siRNA,合成两条互补的寡核苷酸链,退火后连接入pRNATU6.1载体,转化扩增后进行序列测定。用脂质体包裹转染人恶性黑素瘤细胞A375,采用半定量PCR测定HPSE基因RNA水平变化,Western blot检测HPSE蛋白表达的变化。结果:将针对HPSE基因的siRNA的双链寡核苷酸片段克隆到pRNATU6.1载体,经过阳性菌落PCR鉴定与测序,结果正确;转染A375细胞后,半定量PCR和Western blot检测显示,HPSE基因和蛋白的表达水平明显降低(P<0.05)。结论:成功构建了针对HPSE基因的siRNA载体,转染细胞后可抑制HPSE的表达。Objective： To construct heparanase gene-targeted small interfering RNA （siRNA） and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant melanoma A375 cell. Methods： Heparanase gene-targeted hairpin siRNA was designed,two complementary oligonucleotide strand was synthesized and inserted into pRNATU6. 1 vector, which was then identified by PCR and sequencing. Human malignant melanoma A375 cells were transfected with the constructed vector using lipofeetamine method. Semiquantitative PCR was performed to evaluate the heparanase-mRNA expression levels,and Western blot was performed to evaluate the expression of heparanase protein. Results： The vector containing siRNA was identified by PCR and sequencing. the results of semi-quantitative PCR and Western blot showed that the expression levels of both heparanase RNA and protein in transfected A375 cells were decreased significantly（P〈0.05）. Conclusion： The heparanase gene- targeted siRNA and its vector were successfully constructed,which can reduce the heparanase gene and protein expression in transfected cells.

关 键 词：寡核苷酸类 反义 葡糖醛酸糖苷酶 RNA 小分子干扰 RNA干扰 乙酰肝素酶 基因表达 黑色素瘤 

分 类 号：R73-3[医药卫生—肿瘤]