逆转MDR-1小干扰RNA的慢病毒载体系统的构建和鉴定

Construction and identification of the lentiviral vector system expressing MDR1 small interference RNA

目的构建并鉴定表达MDR-1小干扰RNA的慢病毒载体系统并检测其对于喉癌多药耐药细胞系(LSC-1/TAX)多药耐药表型的逆转效果。方法根据MDR-1基因序列,设计3条寡聚核苷酸序列,合成互补DNA链,插入慢病毒表达质粒pLVTHM中,与pCMV-dR8.74和pMD2G共转染293T细胞,包装产生病毒颗粒。以RT-PCR、realtimePCR及Westernblot方法检测三条siRNA在人喉癌耐药细胞系(LSC-1/TAX)中的干扰效率,并采用MTT方法检测干扰前后化疗药物的敏感性。结果通过酶切和测序证实慢病毒载体构建正确,产生能够表达GFP和siRNA的慢病毒颗粒,浓缩滴度为>1×108TU/ml。三条siRNA均可以感染LSC-1/TAX细胞系,其中第三条siRNA敲减效果最佳。结论成功建立逆转MDR-1小干扰RNA的慢病毒载体系统,为逆转喉癌多药耐药表型的实验奠定了基础。

OBJECTIVE To construct the lentiviral vector system expressing MDR1 small interference RNA, and identify reversal efficiency of multidrug resistant phenotype in the human laryngeal cancer multidrug resistance cell lines （LSC-1/TAX）. METHODS Three target sequences of oligonucleotides were selected according to MDR-1 gene sequence, the complementary DNA contained both sense and antisense strands were designed and synthesized. After the oligonucleotides were inserted into the plasmid expression system pLVTHM, the plasmid was cotransfected along with pCMV-dR8.74 and pMD2G into 293T cell lines to package lentiviral particles. Interference efficiency of the lentiviral vector system expressing MDR1 small interference RNA was determined by RT-PCR, real time PCR and Western Blot in the human laryngeal cancer multidrug resistance cell lines （LSC-1/TAX）, drug resistance was measured by MTT assay after interference. RESULTS It was confirmed by digestion and sequencing that lentiviral vector had the correct structure and could express the GFP and siRNA. The functional titer of concentrated virus was more than 1×10^8TU/ml. The vectors expressing 3 target sequences can infect LSC-1/TAX, and the third vector has the best interference efficiency. CONCLUSION The lentiviral vector system expres-sing MDR1 siRNA has been constructed, which is necessary to reverse multidrug resistance phenotype in the human laryngeal cancer multidrug resistance cell lines.