介导MDR1的RNAi腺病毒载体的构建
Construction and Application of Recombinant Adenovirus Vector Expressing the MDR1 shRNA
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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。Abstract: 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 sequence, 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 wasdet...
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