sirna沉默survivin抑制结肠癌细胞株hct116增殖并增强其对奥沙利.doc

RNA干扰GRP78基因肝癌细胞株SMMC-7721增殖 枝江市人民医院 辛祖超陈萌1*，枝江 4432001湖北省第三人民医院 * 通信作者: 陈萌，E-mail: 8364628 @ [摘要] 目的 研究RNA干扰GRP78对肝癌细胞株SMMC-7721增殖和凋亡的影响。方法 将GRP78RNA质粒载体PGRP78转染SMMC-7721，用流式细胞检测转染效率分析细胞周期分布和凋亡，RNA水平检测GRP78效果，MTT 法检测GRP78对SMMC-7721增殖的影响。结果Pla-anti-GRP78转染SMMC-7721细胞48h后，GRP78表达，对照组GRP78 mRNA的表达量为1，RNA组为0.95，而Pla-anti-GRP78组为0.25(P0.05)；Pla-anti-GRP78的SMMC-7721细胞G2期阻滞(55.2%P0.05)；对照组凋亡率为6.6%，RNA组为8.1%，而Pla-anti-GRP78组达58.2%(P0.05)。 结论　RNA干扰GRP78表达抑制SMMC-7721增殖，并诱导凋亡。 [关键词]　 shRNA；GRP78；SMMC-7721细胞 [中图分类号]　R73015 　　[文献标识码]　A 　 sRNA- GRP78 Inhibited Proliferation and Induced apoptosis of liver cancer SMMC-7721 Cells Xin Zhuchao，Yuan Zhengzheng， Zhijiang City Peoples Hospital, Zhijiang 443200 , China Abstract Objective To evaluate the effect of GRP78 special shRNA on proliferation and apoptosis of human liver cancer SMMC-7721 cells. Methods The expression vector of Pla-anti-GRP78 was constructed and transfected into SMMC-7721 by Lipofectin. The protein and mRNA expression of GRP78 was detected by Western blot and real time PCR. The changes of cell cycle after transfection were examined by flow cytometry and MTT assay, respectively. Results In SMMC-7721 cells, the protein and mRNA levels of GRP78 were significantly decreased after transfection, and reduction of proliferation was related to an increase in the fraction of G2 phase. Conclusion The GRP78 special siRNA silenced GRP78, decreased SMMC-7721 cells proliferation and induced their apoptosis. Key words shRNA；GRP78；SMMC-7721 肝癌发生发展是个多阶段多基因参与的过程，与肿瘤细胞增殖凋亡失衡、 基因调节失控密切相关，细胞凋亡受促凋亡因子和抑凋亡因子共同调节。本研究拟用基因重组技术构建的GRP78 特异性的RNA表达载体转染肝癌细胞株SMMC-7721, 研究用RNA 干扰技术下调肝癌细胞GRP78 表达对细胞增殖及凋亡的影响,为肝癌的基因靶向治疗提供理论依据。 1材料和方法 1.1材料　 1.1.1 细胞和质粒 SMMC-7721细胞由本室保存；GRP78特异性干扰质粒Pla-anti-GRP78无关对照质粒命名对照，购自晶赛公司；各质粒均带GFP。 1.1.2 试剂 细胞培养基购自GIBCO公司； Lipofectamine2000和OPTI-MEM培养基购自Invitrogen公司；MTT和DMS0购自上海华舜生物公司；Annexin-v/PI凋亡检测试剂盒购自BD公司； QuantiTect SYBR Green RT-PCR Kit购自Qiagen公司；羊抗人GRP78多克隆抗体购自Santa Cruz Biotechnology公司；HRP标记的兔抗羊二抗购自Amersham公司；β-acti