协和医学杂志

2012, (01) 102-108

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RNA干扰质粒对胰腺癌细胞系Panc-1原癌基因AKT2表达的影响
Effect of RNA Interference Plasmid on the Expression of Oncogene AKT2 in Pancreatic Cancer Cell Line Panc-1

师晓华;梁智勇;吴焕文;任新瑜;刘彤华;
SHI Xiao-hua,LIANG Zhi-yong,WU Huan-wen,REN Xin-yu,LIU Tong-hua Department of Pathology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China

摘要(Abstract):

目的探讨RNA干扰质粒抑制胰腺癌细胞系Panc-1原癌基因AKT2的表达对胰腺癌细胞生长和凋亡的影响,并初步探讨其作用机制。方法选择胰腺癌细胞系Panc-1,构建特异性抑制AKT2表达的RNA干扰质粒,瞬时和稳定转染胰腺癌细胞,采用MTT法及软琼脂克隆形成实验检测胰腺癌细胞生长能力,Heochst染色及Annexin V-FITC/PI染色法检测细胞凋亡情况,通过Westernblot方法检测凋亡蛋白caspase-3表达;并进行裸鼠移植瘤体内转染实验。结果采用RNA干扰质粒沉默胰腺癌细胞系Panc-1原癌基因AKT2,能够有效抑制胰腺癌细胞Panc-1体外生长能力、促进细胞凋亡,诱导凋亡激酶caspase-3的表达;动物体内实验结果显示,干扰质粒能够有效抑制胰腺癌细胞系Panc-1在动物体内的成瘤能力。结论 RNA干扰质粒抑制原癌基因AKT2表达,可有效抑制胰腺癌细胞生长,促进凋亡,针对原癌基因AKT2的基因治疗对胰腺癌具有重要的潜在应用价值。
Objective To explore the effect of RNA interference(RNAi) plasmid on proliferation,apoptosis of status of Panc-1 cells by silencing oncogene AKT2,and investigate its possible mechanism.Methods Pancreatic cancer cell line Panc-1 was applied to constructe the RNAi plasmid-targeting oncogene AKT2 and to transfect cells transiently and stably.The proliferation status was determined using CCK-8 method and soft agar colone formation test.The apotosis status of the cancer cells in vitro was determined using Heochst and Annexin V-FITC/PI methods.The protein level of AKT2 and caspase-3 kinase were detected using Western blotting.Finally,we evaluate the in vivo effect of the recombinant plamids on Panc-1 cells.Results By silencing the oncogenes AKT2,RNAi plasmid effectively down-regulate the mRNA and protein levels of of AKT2 in Panc-1 cells reduced cell proliferation and colony formation of Panc-1 cells,induced apoptosis in Panc-1 cells,increased the protein level of caspase-3 protein,and inhibited tumor growth in vivo.Conclusions RNAi can inhibit the expression of oncogene AKT2 and therefore effectively inhibit the growth of pancreatic cancer cells and promote their apoptosis.Gene therapy targeting AKT2 may be a promising target for pancreatic cancer.

关键词(KeyWords): RNA干扰;胰腺癌;Panc-1细胞系;基因治疗;蛋白激酶B
RNA interference;pancreatic cancer;Panc-1 cell line;gene therapy;protein kinase B

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作者(Author): 师晓华;梁智勇;吴焕文;任新瑜;刘彤华;
SHI Xiao-hua,LIANG Zhi-yong,WU Huan-wen,REN Xin-yu,LIU Tong-hua Department of Pathology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China

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