重组腺病毒介导MRP反义RNA对人肝癌耐药细胞株MDR表型逆转的实验研究  

Reversal of multidrug resistance in human drug-resistant hepatocellular carcinoma cells by recombinant adenoviral delivery of the multidrug resistance-associated protein antisense RNA

作　　者：陈琳[1] 苟兴华 严律南[3] 李德华 韩蕾 赵兰英 胡海洋 

机构地区：[1]南京医科大学第一附属医院普外科,江苏南京210029 [2]成都地奥制药集团有限公司基因工程药物研究室,四川成都610041 [3]四川大学华西医院普外科,四川成都610041

出　　处：《实用肿瘤杂志》2005年第6期484-489,共6页Journal of Practical Oncology

基　　金：国家自然科学基金资助项目(30170925)

摘　　要：目的探讨重组腺病毒载体介导的多药耐药相关蛋白(M RP)反义RNA对多柔比星(阿霉素)诱导的人肝癌耐药细胞株SMM C-7721/ADM多药耐药表型的逆转作用。方法将M RP基因5′端转录起始位点附近长约500 bp的基因片段反向克隆到腺病毒A dE asy系统的穿梭质粒A dT rack-CM V上,通过在细菌内同源重组、并在293细胞中包装、扩增,构建出携带反义M RP的重组腺病毒A dE asy-GFP-A sm rp(简称A d-A sm rp),测定其滴度及对肝癌细胞SMM C-7721/ADM的转染效率;在体外用M O I为100的该重组腺病毒转染肝癌细胞SMM C-7721/ADM,测定转染后细胞对阿霉素(ADM)及柔红霉素(DNR)的半数致死量(IC50)并计算耐药倍数(RF值);在转染后不同时间点(24、48、72、96、120小时)用流式细胞仪动态测定细胞对DNR摄取的变化及细胞膜表面P190表达、并用RT-PCR反映细胞M RP之mRNA水平的变化(以βactin mRNA水平为内对照)。结果重组腺病毒A d-A Sm rp滴度可达2.5×109efu/m l,M O I为100时,即可致90%以上的肝癌细胞SMM C-7721/ADM被感染。体外实验中,该重组腺病毒(M O I=100)转染后的人肝癌耐药细胞SMM C-7721/ADM对ADM、DNR的IC50分别为0.487μg/m l、0.328μg/m l,RF分别为97.4倍、109.3倍,RF分别下降36.8倍和35.4倍。在转染后24小时,M RP之mRNA水平开始下降并呈持续下降趋势(P<0.05),48小时后伴有P190蛋白表达的持续下降(P<0.05),二者趋势一致。转染后48小时,经流式细胞仪测得细胞内DNR浓度出现上升趋势(P<0.05),此后持续明显上升(P<0.05)。结论重组腺病毒介导的M RP反义RNA可有效封闭M RP基因表达,降低P190蛋白水平,在体外实验中能增加人肝癌耐药细胞株SMM C-7721/ADM对化疗药物的敏感性,对肝癌耐药细胞的M DR表型有较好的逆转作用,并为肝癌M DR机制及其逆转方式的研究提供实验基础。Objective To investigate the effect of multidrug resistance-associated protein （MRP） antisense RNA on reversal of the multidrug resistance in human drug-resistant hepatocellular carcinoma （HCC） SMMC-7721/ADM cells. Methods The fragment of MRP gene encoding 5＇region was cloned reversely into the shuttle plasmid. After homologous recombination in bacteria, package and amplification in 293 cells, the recombinant adenoviruses AdEasy-GFP-Asmrp （brief in Ad-Asmrp） containing antisense MRP were obtained. The viral titer and the ability of transduction were measured. Chemosensitivity of all of the cells to ADM and DNR was determined by MTT assay. The levels of the MRP mRNA and its product P190 were measured by RT-PCR and flow cytometry （FCM）,respectively. The accumulation of the DNR in these cells was analyzed by FCM simultaneously. Results Using AdEasy system, the recombinant adenoviruses Ad-Asmrp was successfully constructed. The viral titer was 2. 5 × 10^9efu/ml. More than 90% SMMC-7721/ADM cells could be infected when the MOI was 100. Compared with control groups, IC50 of the cells infected by Ad-Asmrp to adriamycin （ADM） and daunorubicin （DNR） was 0. 487μg/ml and 0. 328μg/ml, respectively, with the factors of resistance （RF） decreased by 36.8- and 35.4-fold,respectively（P〈0.05）. Continuous decrease in levels of MRP mRNA and P190 was observed 24h and 48h after infection,respectively （P〈0. 05）. The intracellular DNR accumulation was increased simultaneously in the Ad-transfectants （P〈0.05）. Conclusion MRP antisense RNA could increase chemosensitivity and partially reverse the MDR of HCC cells in vitro by inhibition of expression of MRP and decrease the levels of P190.

关 键 词：肝肿瘤 RNA 反义 抗药性 多药/遗传学 腺病毒科/遗传学 肿瘤细胞 培养的 重组 遗传 药物耐受性 P-糖蛋白/遗传学 

分 类 号：R735.7[医药卫生—肿瘤]