流式细胞术辅助筛选和纯化人缺氧诱导因子-1α基因沉默肝癌细胞株  

Flow Cytomertry Helps to Screen and Purify the HIF-1α gene Silenced Transfected Hepatoma Carcinoma Cells

作　　者：王燕[1] 李志伟[2] 楚青[2] 江萍[3] 危群[1] 陆晓青[2] 

机构地区：[1]昆明医学院第二附属医院病理科,云南昆明650101 [2]昆明医学院第二附属医院中心实验室,云南昆明650101 [3]昆明医学院病理教研室,云南昆明650031

出　　处：《昆明医学院学报》2011年第4期20-25,共6页Journal of Kunming Medical College

基　　金：云南省应用基础研究基金资助项目(2005C0040Q)

摘　　要：目的利用流式细胞术(FCM)协助筛选、纯化RNA干扰(RNAi)效率高的人缺氧诱导因子1-α(HIF-1α)基因沉默肝癌细胞株.方法选择带Neor及GFP基因的质粒pGenesil-1.1构建HIF-1α靶向小干扰RNA(siRNA)重组表达载体,转染人肝癌细胞株SMMC7721.首先用G418筛选,然后用FCM检测出常氧、缺氧条件下HIF-1α蛋白表达抑制效率最高的转基因细胞株.根据FCM可分选GFP阳性细胞这一特点,进一步用流式细胞分选仪分选纯化转基因细胞,最后用PCR和免疫组化方法鉴定纯化细胞株的HIF-1α干扰效率.结果单用G418筛选HIF-1α干扰组细胞最高筛选效率为32.9%,FCM检测HIF-1α蛋白抑制率为78.7%～92.8%;合并使用G418和FCM分选纯化后,最佳HIF-1α干扰组筛选效率达94.4%,常氧、缺氧条件下对HIF-1αmRNA表达抑制率分别为95.5%、85.2%.结论合并使用G418及FCM分选纯化得到了高RNAi效率的人HIF-1α基因沉默肝癌细胞株,为进一步研究HIF-1α与肝癌关系及靶向HIF-1α的肝癌基因治疗提供了实验基础.Objective To screen and purify highly efficient gene silencing human hepatoma carcinoma cell lines targeting HIF-1α gene by use of flow cytometry.Methods pGenesil-1.1 plasmid containing both Neor and GFP genes was selected.After the plasmids had been transfected into SMMC7721 cells,the transgenic cells were selected by G418 firstly,and then detected the expression of HIF-1α protein under normoxia or hypoxia condition by FCM,so the most efficient transgenic cell lines inhibiting HIF-1α protein expression were selected.According to GFP-positive cells separated by FCM,and the most efficient RNA interfere transgenic cell lines were separated and purified by FCM,and the interference efficiency were identified by RT-PCR and immunohistochemistry staining methods finally.Results The highest purity of GFP positive cells was 32.9% in HIF-1α interfere groups when selected by G418 alone,the inhibition ratio of HIF-1α protein was from 78.7% to 92.8% detected by FCM.However,the purity of GFP positive cells was 94.4% in the optimal group of HIF-1α interference sorting and purifying by both G418 and FCM,and the inhibition ratio on HIF-1α mRNA expression were 95.5% and 85.2% respectively under normoxia or hypoxia condition.Conclusions The highest efficiency of RNAi transgenic human hepatoma carcinoma cell line targeting HIF-1α can be sorted and purified by combination of G418 and FCM.This study lays a foundation for further study on the relationship between HIF-1α and hepatoma carcinoma,and the gene therapy for hepatoma carcinoma targeting HIF-1α.

关 键 词：缺氧诱导因子-1a RNA干扰 流式细胞术 

分 类 号：R735.3[医药卫生—肿瘤]