循环包装回收技术在筛选肝癌细胞相关耐药基因中的应用

Screening of drug resistent gene by cyclical packaging rescue of hepatocellular carcinoma retroviral cDNA libraries

摘要:

肝癌先天性多表达多药耐药基因，严重影响肝癌的化疗效果，筛选肝癌细胞中的耐药基因，研究其耐药机制有助于提高肝癌化疗效果，提高肝癌的治愈率。首先构建肝癌细胞逆转录病毒的cDNA文库，感染成纤维细胞，使得逆转录病毒基因整合进细胞，加药筛选，存活细胞中的基因再次包装成病毒，用于下一轮筛选。采用循环包装回收 (Cyclical packaging rescue，CPR) 技术进行肝癌细胞耐药基因的筛选即是通过病毒包装将基因从细胞中钓取出来，相比于常规筛选方法，仅通过一轮筛选可能会出现很多假阳性基因，采用CPR技术则是通过多轮筛选，很大程度减少了假阳性细胞的出现。通过该方法经过四轮筛选获得核糖体蛋白S11 (RPS11)、核糖体蛋白L6 (RPL6)、核糖体蛋白L11 (RPL11)、核糖体蛋白L24 (RPL24) 等几种基因，经初步检测，增加了细胞的耐药性。

Abstract:

Multidrug resistant genes are highly expressed in hepatocellular carcinoma that seriously affects the effect of chemotherapy. Screening of resistant genes from HCC cells and studying its mechanism of drug resistance will be helpful to improve the effecacy of chemotherapy for hepatocellular carcinoma. Here we described an alternative method called cyclical packaging rescue (CPR). First we constructed a retrovirus cDNA library of hepatoma cells and used it to infect fibroblasts. Then we added drugs to screen survival cells. The survival cells, stably integrated helper-free retroviral libraries, were recovered rapidly after transfection with plasmids expressing retroviral gag-pol and env genes. Through this method, retroviral RNAs were directly repackaged into new infectious virions. Recovered retroviral supernatant was then used to reinfect fresh target cells. When performed in concert with selection using functional assays, cDNAs regulating functional responses could be identified by enrichment through multiple rounds of retroviral library recovery and retransmission. Using CPR, we obtained several cDNAs. After a preliminary detection, we found Ribosomal protein S11 (RPS11), Ribosomal protein L6 (RPL6), Ribosomal protein L11 (RPL11), Ribosomal protein L24 (RPL24) possibly had drug resistant function.