HCV发夹状siRNA表达质粒的构建及作用研究  

Construction of HCV hairpin siRNA expression vector and the effects

作　　者：陈佳玉[1] 戚永孝[1] 王泰鑫[2] 张丽婷[1] 林平[1] 

机构地区：[1]台州学院医学院,浙江台州318000 [2]海盐县人民医院,浙江嘉兴市海盐314300

出　　处：《中国热带医学》2013年第5期546-547,594,共3页China Tropical Medicine

基　　金：浙江省自然科学基金(No.Y2100248);浙江省科技厅项目(No.2009C33155);浙江省卫生厅项目(No.2009A218);台州市科技局项目(No.102KY15);浙江省中医药管理局项目(No.2011ZA113)

摘　　要：目的建立HCV感染动物模型,并对HCV进行RNA干涉。方法制备HCV感染的HepG2细胞,RT-PCR法鉴定后腋下注射裸鼠,免疫组化法检测瘤组织内HCV NS3表达情况。制备带绿色荧光蛋白(GFP)的HCVsiRNA表达质粒,转染HepG2细胞,观察GFP表达情况。将此质粒注射模型小鼠,取瘤组织,RT-PCR及免疫组化法对其进行检测。结果在HCV感染的裸鼠实体瘤中检测到HCV NS3蛋白的稳定表达。成功构建了HCV发夹siRNA表达质粒,该质粒转染细胞后能够在其内正确表达。将该质粒尾静脉注射HCV感染的模型小鼠,注射4d后瘤组织内HCV NS3抗原阳性细胞阴转。结论成功建立了HCV感染的裸鼠模型,构建了HCV发夹siRNA表达质粒,该质粒能成功地干涉模型小鼠中的HCV。Objective To establish HCV infection animal model,and silence the HCV in it.Methods HCV infected HepG2 cells were prepared,injected them into nude mice at armpit after identified with RT-PCR assay and analyzed the HCV NS3 protein expression level in these tumor tissues by immunohistochemical assay.HCV hairpin siRNA expressional plasmid was constructed with a green fluorescent protein gene（GFP）as marker.Transfected this expression plasmid into HepG2 cells,and observed the expression level of GFP.This plasmid was injected into the mouse model through tail vein,got the tumor tissues and detected it with RT-PCR and immunohistochemical methods.Results HCV NS3 protein was detected stably expressing in solid tumors of nude mice.The HCV hairpin siRNA expression plasmid was constructed successfully,and it could express correctly after transfected into cell.Injected this plasmid into the HCV infected mice model through caudal vein,4 days later,HCV NS3 antigen positive cells in tumor tissue was changed into negative.Conclusions HCV infected nude mice model was established successfully,the HCV hairpin siRNA expression plasmid was constructed and it can interfere the HCV successfully in the mice model.

关 键 词：丙型肝炎病毒 RNA干涉 感染模型 

分 类 号：R512.63[医药卫生—内科学]