SiRNA稳定靶向干扰HDGF肺腺癌细胞株的建立及干扰效率检测  

Establishment of a lung adenocarcinoma cell line stably expressing small interfering RNA targeting hepatoma-derived growth factor and detection of interference effect

作　　者：孟婕[1] 曹立明[1] 胡成平[1] 郑智远[1] 

机构地区：[1]中南大学湘雅医院呼吸科,湖南长沙410008

出　　处：《南方医科大学学报》2009年第11期2233-2236,共4页Journal of Southern Medical University

基　　金：湖南省科技计划项目(2007FJ4159)

摘　　要：目的构建针对肝癌衍生生长因子(HDGF)基因的siRNA表达载体,建立稳定干扰HDGF基因表达的肺腺癌细胞株,检测干扰效率。方法实时荧光定量PCR比较肺腺癌细胞株SPC-A-1、10例肺腺癌组织和其配对的癌旁肺组织HDGF基因表达差异。构建shRNA-HDGF慢病毒表达载体,测序鉴定序列的正确性。随后用脂质体的方法将载体转染入肺腺癌细胞株SPC-A-1中,经杀稻瘟菌素筛选后,稳定表达siRNA-HDGF的细胞株单克隆细胞株建立。实时荧光定量PCR检测干扰效率,筛选干扰效率最高的细胞株。结果HDGF基因在腺癌细胞株SPC-A-1和肺腺癌组织明显高表达。测序证实,构入慢病毒载体中shRNA序列正确。一共筛选了5个siRNA-HDGF细胞株。与对照载体和单纯细胞株相比,最高干扰HDGF表达的效率为75%。结论HDGF基因在肺腺癌细胞株及肺癌组织中高表达;针对HDGF的siRNA慢病毒表达载体成功构建;在其导入肺腺癌细胞株后能稳定干扰HDGF基因的表达。Objective To construct a small interfering RNA（siRNA） expression vector targeting hepatoma-derived growth factor（HDGF） and establish a lung adenocarcinoma cell line stably expressing siRNA-HDGF.Mehtod RT-PCR was used to examine HDGF expression in lung adenocarcinoma samples and the matched adjacent lung tissues,and also in lung adenocarcinoma SPC-A-1 cell line.A recombinant lentivirus shRNA-HDGF vector was constructed and transfected into SPC-A-1 cells via Lipofectamine 2000,and the cells with stable expression of HDGF-siRNA was screened by blasticidin selection.The interference effect of siRNA-HDGF was assessed by real-time PCR.Results Compared to the adjacent lung tissues,lung adenocarcinoma and SPC-A-1 cells showed increased expression of HDGF.The recombinant lentivirus shRNA-HDGF vector was successfully constructed and verified by sequence analysis.siRNA-HDGF recombinants markedly inhibited the expression of HDGF in SPC-A-1 cells.Conclusion HDGF expression increases in lung adenocarcinoma and SPC-A-1 cell lines.The recombinant siRNA-HDGF lentivirus vector can inhibit the expression of HDGF in SPC-A-1 cells.

关 键 词：肝癌衍生生长因子 SIRNA 肺肿瘤 

分 类 号：R734.2[医药卫生—肿瘤] R392.11[医药卫生—临床医学]