ITGA5慢病毒shRNA表达载体及Bel-7404稳定细胞系的构建  

Construction of shRNA Lentiviral Expression Vector of ITGA5 Gene and Establishment of Its Stable Transfection Bel-7404 Human Hepatoma Carcinoma Cell Line

作　　者：郭亚妹 孙昊[3] 何彩霞 杨小利 杨少奇 GUO Yamei;SUN Hao;HE Caixia;YANG Xiaoli;YANG Shaoqi(Ningxia Medical University,Yinchuan 750004;Department of Gastroenterology,the General Hospital of Ningxia Medical University,Yinchuan 750004;China Petroleum Center Hospital,Langfang 065000;Department of Gastroenterology,People＇s Hospital of Ningxia Autonomous Region,Yinchuan 750002)

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学总医院消化内科,银川750004 [3]中国石油中心医院,廊坊065000 [4]宁夏回族自治区人民医院消化内科,银川750002

出　　处：《宁夏医科大学学报》2018年第3期249-253,260,共6页Journal of Ningxia Medical University

基　　金：宁夏医科大学优势学科群建设项目(2001060703)

摘　　要：目的构建整合素α5基因(ITGA5)的短发卡RNA(shRNA)慢病毒表达载体,并建立稳定沉默ITGA5表达的肝癌细胞系Bel-7404。方法设计3条ITGA5基因特异性shRNA靶序列和1条非特异性序列作为阴性对照(NC),分别克隆到GV493质粒载体中,转化感受态细胞后挑取阳性克隆子进行DNA测序鉴定,包装成重组慢病毒,并纯化、测定病毒滴度。将4组慢病毒载体分别感染人肝癌细胞Bel-7404,使用嘌呤霉素筛选后,在荧光显微镜下观察GFP的表达,q PCR和Western blot分别检测各组中ITGA5的基因和蛋白质表达水平。结果 ITGA5基因shRNA慢病毒表达载体经过测序鉴定构建成功,q PCR和Western blot检测结果显示3种慢病毒干扰组的ITGA5表达水平均低于NC组(P均<0.05)。NC组m RNA的表达量为(100±2.3)%,ITGA5-shRNA-1、ITGA5-shRNA-2、ITGA5-shRNA-3组m RNA相对表达量分别为(10.7±3.7)%、(16.7±1.5)%、(11.8±2.9)%,且其干扰效率均达到70%以上,可选择其中一个干扰靶点进行后续实验。结论成功构建ITGA5基因shRNA慢病毒表达载体,感染肝癌Bel-7404细胞并成功筛选出稳定沉默细胞系,为进一步研究ITGA5在肝癌中的生物学功能和作用机制提供了实验基础。Objective To construct a short hairpin RNA（shRNA）lentiviral vector expressing the integrin alpha 5 gene（ITGA5） and stably knock down ITGA5 in human hepatoma carcinoma cell line Bel-7404.Methods Three specific shRNA sequences and one nonspecific sequence（used as negative control,NC）were designed and synthesized.They were cloned into the GV493 plasmid vector and transformed into competent cells.Then the positive clones were chosen for DNA sequencing. The recombinant lentiviral vector was packaged and purified,and the virus titer was assayed. Then Bel-7404 hepatoma carcinoma cell was infected with the four groups of lentivirus.After screening with puromycin,the expression of GFP was observed under the fluorescence microscope,and the expression level of ITGA5 m RNA and protein in each group was detected by q PCR and Western blot. Results shRNA lentiviral expression vector targeting ITGA5 gene was confirmed successfully by DNA sequencing,q PCR and Western blot showed that the ITGA5 expression level of each experimental group was significantly lower in group NC,and the differences were statistically significant（P0.05）. The m RNA expression of NC group,and the ITGA5-shRNA-1,ITGA5-shRNA-2,ITGA5-shRNA-3 groups were（100±2.3）%,（10.7±3.7）%,（16.7±1.5）%,（11.8±2.9）%,and its interference efficiencies were all more than 70%,so one of the target can be selected for follow-up experiments. Conclusion The ITGA5-shRNA lentiviral expression vector was successfully constructed and stably infected into human hepatoma carcinoma cell line Bel-7404.The result will provide experimental basis for further study on the biological function of ITGA5 in hepatocellular carcinoma.

关 键 词：整合素α5基因 短发卡shRNA 慢病毒表达载体 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]