短发卡状RNA对AKT2基因表达的抑制诱导人胰腺癌细胞凋亡  

Apoptosis was Induced in Human Pancreatic Cancer Cells Using Short Hairpin RNA Interfering AKT2 Kinase Expression

作　　者：蔡庆和[1] 陈先祥[1] 张征[2] 王江华[2] 车军[1] 

机构地区：[1]郧阳医学院附属人民医院普外科,湖北十堰442000 [2]湖北中医学院,湖北武汉430060

出　　处：《郧阳医学院学报》2006年第4期198-201,204,F0002,共6页Journal of Yunyang Medical College

摘　　要：目的：构建AKT2基因特异性短发卡状RNA（shRNA）真核表达载体，运用RNA干扰（RNAi）技术，观察其对人胰腺癌细胞SW1990和Capan2中AKT2蛋白激酶表达的抑制作用，并探讨其增强肿瘤细胞对化疗药物敏感性，促进细胞凋亡的机制。方法：运用基因工程技术，设计合成针对AKT2 mRNA的特异性shRNA，构建真核表达载体pAKT2-shRNA。将pAKT2-shRNA经脂质体包裹转染人胰腺癌细胞SW1990和Capan2，荧光显微镜观察细胞内红色荧光蛋白的表达，计算转染效率，RT-PCR、Western Blot检测AKT2的表达及干扰效率，FACS检测细胞凋亡率来比较转染前后人胰腺癌细胞的抗凋亡能力。结果：①经双酶切及测序证实pAKT2-shRNA构建成功。②SW1990和Capan2细胞转染pAKT2-shRNA后，荧光显微镜下观察，发红色荧光的细胞占总细胞数的35％～40％；RT-PCR和Western Blot检测结果示，转染后AKT2 mRNA和蛋白表达显著降低。③FACS检测结果示，转染pAKT2-shRNA后，紫杉醇30 nM作用24h，与对照组相比，细胞凋亡率显著升高（P〈0．05）。结论：pAKT2-shRNA能有效抑制胰腺癌细胞AKT2基因表达，增强肿瘤细胞对化疗药物的敏感性，促进肿瘤细胞凋亡。Objective To construct the short hairpin RNA （shRNA） eukaryotic expression vector specific to human AKT2 gene （pAKT2- shRNA） to investigate its silencing effect on AKT2 kinase and the mechanism of the induced cell apoptosis. Methods ShRNA targeting AKT2 mRNA was designed and synthesized and pAKT2 -shRNA eukaryotic expression vector was constructed using gene engineering technique. Lipofectamine^TM 2000 transfection system was used to transfect pAKT2 - shRNA into human pancreatic cancer cell lines SW1990 and Capan2. The efficiency of transfection was evaluated by immunofluoreseence microscope, and the expression change of AKT2 was examined by semi - quantitative reverse transcription polymerase chain reaction （ RT - PCR） and western blot. The apoptosis induced by pAKT2 - shRNA was analyzed by FACS. Results pAKT2 - shRNA plasmid was successfully constructed. AKT2 expression in SW1990 and Capan2 cells were obviously inhibited by pAKT2 - shRNA. FACS analyses showed that, compared with control group, pAKT2 - shRNA increased apoptosis of SW1990 and Capan2 cells induced with paclitaxel. Conclusion pAKT2 - shRNA significantly inhibited AKT2 expression at mRNA and protein level and increased apoptosis of human pancreatic careinoma cells induced with paclitaxel. The successful tranfection of pAKT2 - shRNA in human pancreatic carcinoma cells may enhance the therapeutic effect of chemotherapeutics.

关 键 词：AKT2 SHRNA 胰腺癌 细胞凋亡 

分 类 号：R735.9[医药卫生—肿瘤]