稳定干扰核糖体蛋白RPS7基因表达的宫颈癌HeLa细胞株的建立  

Establishment of HeLa Cervical Cancer Cell Linewith Stable Knock- down of RPS7

作　　者：丁慧[1] 禹立霞[1] 凌静娴[2] 刘宝瑞[1] 张葵[3] 王庆飞[3] 

机构地区：[1]南京大学医学院附属鼓楼医院肿瘤科,南京市210008 [2]南京大学医学院附属鼓楼医院妇科,南京市210008 [3]南京大学医学院附属鼓楼医院检验科,南京市210008

出　　处：《医学分子生物学杂志》2013年第6期318-322,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81302241）,南京市医学科技发展专项

摘　　要：目的建立稳定抑制RPS7基因表达的宫颈癌HeLa细胞株。方法设计并合成靶向人RPS7基因的shRNA寡核苷酸片段，克隆到逆转录病毒载体pSIREN中，构建重组质粒pSIREN-RPS7-shRNA，转染293T细胞，将包装产生的重组逆转录病毒感染宫颈癌HeLa细胞，经嘌呤霉素筛选获得稳定的细胞克隆，用real-timePCR和Western印迹检测细胞中RPS7mRNA和蛋白表达水平。结果获得了经测序鉴定正确的重组逆转录病毒质粒，逆转录病毒感染HeLa细胞后用嘌呤霉素筛选出的稳定细胞中，RPS7mRNA和蛋白水平均显著低于干扰对照细胞。结论成功构建了靶向人RPS7基因的shRNA逆转录病毒载体，建立了稳定抑制RPS7基因表达的宫颈癌HeLa细胞株．为进一步研究RPS7在宫颈癌中的生物学功能和作用机制提供了可靠的细胞模型。Objective human ribosomal protein S7 To establish a HeLa cervical cancer cell line with stable knockdown of gene （RPS7） . Methods shRNA sequences targeting RPS7 were de- signed and cloned into the retrovirus vector pSIREN to obtain the recombinant plasmids pSIREN- RPS7-shRNA. The recombinant plasmids were thereafter transfected into 293T cells for virus pack- age, which was followed by infection of HeLa cells with the packaged virus. Then puromycin was used to generate the stable virus-infected HeLa cells. The RPS7 gene expression level was measured by real-time PCR and Western blotting. Results Recombinant retrovirus shRNA plasmids were cor- rectly constructed as confirmed by sequencing results. RPS7 mRNA and protein levels were signifi- cantly decreased in puromycin-treated pSIREN-RPS7-shRNA- transfected HeLa cells compared with control cells. Conclusions The recombinant retrovirus shRNA vectors targeting RPS7 were success- fully constructed and HeLa cell models with stable knockdown of RPS7 were established, which pro- vide a useful cell line model to further investigate the biological roles of RPS7 and its action mecha- nisms in cervical cancers.

关 键 词：RPS7基因 SHRNA 逆转录病毒载体 宫颈癌 

分 类 号：R737.33[医药卫生—肿瘤]