摘要
目的介导MDR1的RNAi腺病毒载体,探讨RNA干扰MDR1基因对人肝癌细胞的作用。方法根据MDR1mRNA序列构建表达MDR1mRNA特异的shRNA的腺病毒穿梭质粒pshuttle-MDR1。与腺病毒载体体内重组为pAd-MDR1后转染人肝癌细胞SMMC-7721,以FCM检测细胞表面膜蛋白P-gp表达阳性率,以共聚焦显微镜检测细胞内Rh123的潴留,WesternBlot检测P-gp蛋白量的变化。结果构建成pshuttle-MDR1经限制性酶切和PCR证实与设计一致,将pAd-MDR1转染肝癌细胞SMMC-7721后,FCM检测细胞表面膜蛋白P-gp表达阳性率为22.9%和30.8%,远低于对照组(85.8%)。WesternBlot经病毒感染的SMMC-7721/R的P-gp含量明显低于对照SMMC-7721/R,而与SMMC-7721/S细胞接近。结论成功构建了pshuttle-MDR1腺病毒载体,并有效干扰了肝癌细胞细SMMC-7721MDR1的表达。
Objective To construct a recombinant adenovirus vector expressing the MDR1 shRNA and investigate its effect in human hepatocellular carcinoma cell. Methods According to the MDR1 mRNA se quence, the DNA segment homogenizing wish MDR1 shRNA was synthesized and cloned into the shuttle plasmid pshuttle-MDR1. The later and adenovirus vecter were cotransfected to package the pAd-MDR1. pAd-MDR1 was transfected into human hepatocellular carcinoma cell SMMC-7721. The expression of P-gp on the cellular membrane wasdetected by FCM and western blot. Rhodamine123 （Rh123） retention in SMMC-7721 cell was detected by confocal microscopy. Results The pshuttle-MDR1 plasmid was identified with the method of PCIL After transfecting SMMC-7721 cell with pAd-MDR1, FCM detected that the positive rate of P-gp expression（22. 9% and 30. 8%） was far lower than control （85. 8%）, and in SMMC-7721/R cell, western blot detected that the quantity of P-gp was lower than control, just like the quantity in SMMC-7721/S cell. Conclusion The pshuttle-MDR1 adenovirus vector is prepared successfully and its inhibition of MDR1 in SMMC-7721 cell is confirmed.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2006年第11期798-801,共4页
Cancer Research on Prevention and Treatment
基金
湖北省教育厅资助项目(2004D006)
关键词
RNAI
肝癌细胞
多药耐药基因
腺病毒载体
RNA interference
Hepatocellular carcinoma cell
MDR1
Adenovirus vector
