刘瑶,石祥,方文
LIU Yao,SHI Xiang,FANG Wen

• 1:贵州医科大学
• 2:贵州医科大学临床生化教研室

摘要(Abstract)：

目的:筛选不均一核糖核蛋白A2/B1(hnRNPA2/B1)的短发夹RNA(shRNA)慢病毒载体感染宫颈癌细胞株Hela细胞的稳定细胞株。方法:利用hnRNPA2/B1 shRNA慢病毒载体转染Hela细胞,采用嘌呤霉素抗性方法筛选稳定转染的Hela细胞系;将Hela细胞分为空白组(Hela)、阴性组(NC-shRNA)、干扰组1(shRNA1)、干扰组2(shRNA2)、干扰组3(shRNA3)和干扰组4(shRNA4),实时荧光定量PCR(RT-PCR)检测各组细胞hnRNPA2/B1 mRNA表达水平,Western blot检测hnRNPA2/B1蛋白表达水平,筛选干扰沉默效率最高的细胞作为稳定沉默hnRNPA2/B1基因的稳转株。结果:经筛选获得稳定转染的宫颈癌Hela细胞株,干扰组2,干扰组3和干扰组4与空白组比较,hnRNPA2/B1基因和蛋白表达量降低,差异有统计学意义(P<0.05),其中干扰组4沉默效率最高。结论:成功构建hnRNPA2/B1 shRNA慢病毒载体感染的Hela稳定细胞株。
Objective: To screen the stable Hela cell line infected with hnRNPA2 /B1 shRNA lentivirus vector. Methods: hnRNPA2 / B1 shRNA lentivirus vector was used to stably transfect into He La cells. The Hela cell lines were screened by using the method of resistance of puromycin. Hela cell lines were divided into blank group( Hela group),negative group( NC-shRNA group),interference group1( shRNA1group),interference group2( shRNA2 group),interference group3( shRNA3 group)and interference group4( shRNA4 group). RT-PCR was adopted to detect the mRNA expression level of hnRNPA2 / B1 and Western blot adopted to detect the protein expression level of hnRNPA2 / B1. A group of cells with the highest silencing efficiency was selected as the Hela lines of hnRNPA2 / B1 gene stable silencing. Results: Stably transfected He La cell line of cervical cancer was obtained by screening. Compared with blank group,mRNA level and protein level of hnRNPA2 / B1 gene expression significantly decreased in shRNA2 group,shRNA3 group and shRNA4 group( P < 0. 01). Among them,silence efficiency was highest in shRNA4 group. Conclusion: Stable Hela cell line infected with hnRNPA2 / B1 shRNA lentivirus vector is successfully constructed.

关键词(KeyWords)： RNA干扰;短发夹RNA;宫颈癌;Hela细胞;慢病毒;不均一核糖核蛋白基因
RNA interference;Short hairpin RNA;cervical carcinoma;Hela cell line;lentivirus;heterogeneous nuclear ribosomal protein gene

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81560481)

作者(Author): 刘瑶,石祥,方文
LIU Yao,SHI Xiang,FANG Wen

DOI: 10.19367/j.cnki.1000-2707.2016.06.002

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