超声靶向辐照微泡造影剂介导重组腺相关病毒转染肾癌细胞  

Ultrasound targeted microbubbles destruction technology mediated rAAV transfection in renal carcinoma cells

作　　者：李凡[1] 杜联芳[1] 王惠萍[2] 韦芳[2] 

机构地区：[1]上海交通大学医学院附属第一人民医院超声科,上海200080 [2]上海交通大学医学院附属第一人民医院中心实验室,上海200080

出　　处：《中国医学影像技术》2011年第5期895-900,共6页Chinese Journal of Medical Imaging Technology

基　　金：国家自然科学基金(30772369);国家自然科学基金青年科学基金(8100068)

摘　　要：目的以肾癌细胞为研究对象,探索超声靶向破坏微泡（UTMD）技术对重组腺相关病毒（rAAV）转染率的影响。方法应用不同感染复数（MOI）的rAAV2转染786-0细胞观察转染率。用不同条件UTMD预辐照rAAV2,观察病毒活性。以不同条件UTMD联合rAAV2转染人肾透明细胞癌（786-0）细胞,测定转染率及细胞生存率,RT-PCR检测细胞内病毒载体基因拷贝数。结果 1×10^4-1×10^6MOI的rAAV2在786-0细胞的转染率为（17.28±2.44）%。UTMD声强≤2.0 W/cm2,时间≤120 s,微泡体积比≤40%,频率1 MHz,占空比（DC）50%,脉冲重复频率（PRF）100 Hz时,UT-MD辐照不影响rAAV2的转染活性。在不影响细胞生存率及rAAV2活性的参数下（声强1 W/cm2,时间60 s,微泡体积比20%,频率1 MHz,DC 50%,PRF 100 Hz）,UTMD介导rAAV2组细胞的转染率较单纯rAAV组提高2~3倍,并在5天内稳定维持提高效应;实时PCR显示,UTMD介导rAAV2组细胞内病毒载体量较单纯rAAV组提高近9倍。结论 UTMD可安全、有效且稳定地提高rAAV2在肾癌细胞的转染率。Objective To investigate whether ultrasound targeted microbubble destruction（UTMD） technology can enhance recombinant adeno-associated virus（rAAV） transduction efficiency in human renal carcinoma cells（786-0）.Methods Different multiplicity of infections（MOIs） of rAAV2 transduction efficiency in 786-0 cells was investigated.rAAV2 was pre-exposured to determine whether its infection function was destroyed with this technology.Then combining with different conditions of UTMD technology,the transduction efficiency of rAAV2 and the cell viability in 786-0 cells were analyzed.The virus genome copies were analyzed with RT-PCR.Results The average transduction efficiency of rAAV2 in 786-0 cells at MOIs of 1×104～1×106 was（17.28±2.44）%.When acoustic intensity ≤2.0 W/cm2,exposure time ≤120 s,MBs volume ratio ≤40%,frequency was 1 MHz,duty cycle（DC） was 50% and pulse recurrence frequency（PRF） was 100 Hz,UTMD pre-exposure could not damage the infectious activity of rAAV2.Under optimized conditions of UTMD（acoustic intensity 1.0 W/cm2,exposure duration 60 s,MBs volume ratio 20%,frequency 1 MHz,DC 50%,PRF 100 Hz）,the efficiency of rAAV2 was enhanced nearly 2—3 times than rAAV2 without UTMD and the cell viability did not significantly decreased.The increasing effect maintained continuously in 5 days.Real-time PCR showed the genomic DNA copy of virus vector enhanced 9 times when rAAV2 combined with UTMD technology.Conclusion UTMD technology can enhance rAAV transduction efficiency in less permissive renal carcinoma cells safely,effectively and stably.

关 键 词：超声学 微泡 腺病毒科 依赖病毒 细胞 

分 类 号：R445.1[医药卫生—影像医学与核医学]