慢病毒靶向携带Akt1 shRNA对胃癌细胞SGC―7901凋亡和增殖影响.docVIP

慢病毒靶向携带Akt1 shRNA对胃癌细胞SGC―7901凋亡和增殖影响 　　[摘要] 目的 探讨慢病毒载体靶向携带Akt1基因短发夹RNA（Akt1-shRNA）对胃癌细胞SGC-7901凋亡和增殖的影响。 方法 构建表达Akt1-shRNA的慢病毒载体，转染293T细胞后获得Akt1-shRNA的慢病毒颗粒，病毒感染胃癌SGC-7901细胞（SGC-LV-shAkt1）为实验组细胞，SGC7901细胞和转染慢病毒空载体SGC7901细胞（SGC-LV-shCON）为对照组细胞。Real-Time PCR检测Akt1 mRNA表达水平，Western blot检测胃癌细胞Akt1、phospho-Akt（ser473）和bcl-2蛋白表达水平，然后流式双染法、生长速率等方法检测Akt1 shRNA对SGC-7901细胞凋亡及增殖能力的影响。 结果 成功构建Akt1基因RNAi的慢病毒载体LV-shAkt1，病毒滴度为5×108 TU/mL。SGC-7901细胞转染后，实验组（SGC-LV-shAkt1）Akt1 mRNA 和Akt1、p-Akt、bcl-2蛋白表达水平较对照组（SGC-7901、SGC-LV-shCON）降低（P 0.01）；（38.7±3.5）%的实验组细胞凋亡（P 0.05）。SGC-LV-shAkt1细胞生长曲线平缓，生长速度较另两种细胞（SGC-7901、SGC- LV-shCON）明显降低。 结论 靶向Akt1的RNAi 能有效抑制胃癌细胞SGC-7901的生长，诱导胃癌细胞凋亡，其机制可能与抑制PI3K/Akt信号通路，下调抗凋亡蛋白bcl-2有关。 　　[关键词] Akt1；慢病毒载体；胃癌；凋亡 　　[中图分类号] R735.2 [文献标识码] A [文章编号] 1673-7210（2017）06（b）-0023-05 　　Department of Gastroenterology， Renmin Hospital of Wuhan University Hubei Key Laboratory of Digestive System Disease， Hubei Province， Wuhan 430060， China 　　[Abstract] Objective To investigate the apoptosis and proliferation effect of lentiviral vectors shRNA for Akt1 to human gastric carcinoma SGC-7901 cells. Methods The lentiviral vectors Akt1-shRNA were constructed， lentivirus Akt1-shRNA was transfected into human gastric carcinoma SGC-7901 cells， the SGC-7901 cell strained with Akt1 gene silencing perpetually （SGC-LV-shCON） was as conrol group cell and amplified （SGC LV-shAkt1） was as experiment group cell. Effect of Akt1 on SGC-7901 cells were measured by RT-PCR， Western-blot， flow cytometry of annexin V-FITC/PI， tumor growth curve. Results Akt1 RNAi lentiviral vectors LV-shAkt1 was established successfully， virus titer was 5×108 TU/mL. After SGC-7901 cell transfection， the expression of Akt1 mRNA in experiment group （SGC- LV-shAkt1） was weaker than that in the control group （SGC-7901， SGC- LV-shCON）， and Akt1， p-Akt and bcl-2 protein expressions in experiment group was inhabited （P 0.01）， （38.7±3.5）% cell in experiment group happened apotosis （P 0.05）. The cell growth curve