病毒与非病毒载体转染新生大鼠血管纹边缘细胞的比较研究  

Comparison of gene transfer into newborn rat cochlear marginal cells in primary culture using different vectors

作　　者：杨阳[1] 孔维佳[1] 李隽[1] 胡钰娟[1] 钟毅[1] 郝亚楠[1] 赵学艳[1] 彭炜[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院耳鼻咽喉-头颈外科,武汉430022

出　　处：《中华耳科学杂志》2008年第2期221-227,共7页Chinese Journal of Otology

基　　金：国家自然科学基金重点项目(No.30730094);国家科技部十一五科技攻关(No.2007BAI18B13);国家杰出青年基金资助项目(No.39925035)

摘　　要：目的通过病毒与非病毒载体对原代培养的新生大鼠内耳血管纹边缘细胞转染情况的比较,选择出适合边缘细胞的基因治疗载体。方法用质粒(pEGFP-N1)、血清5型重组腺病毒(rAd5)以及血清2型重组腺相关病毒(rAAV2)三种载体转染新生大鼠(≤72小时)耳边缘细胞,通过对转染效率、细胞凋亡以及细胞活性的检测将三种载体加以比较。结果对边缘细胞的转导效率依次为rAd5>rAAV2>pEGFP-N1,AnnexinⅤ-APC/7-AAD双染法检测转染阳性细胞的损伤情况依次为rAAV2<rAd5<pEGFP-N1,CCK-8法检测转染后的细胞活性依次为rAAV2>rAd5>pEGFP-N1。结论病毒载体相对于非病毒载体以其高转导效率、低细胞毒性在对原代培养的新生大鼠耳边缘细胞的转染中表现出明显优势。与血清2型重组腺相关病毒比较,相对高效、低毒性的血清5型重组腺病毒更适用于原代培养的新生大鼠血管纹边缘细胞的基因转染研究。Objective Plasmids, recombinant adenoviruses （rAds）, and adeno-associated viruses （rAAVs） are frequently used vectors, each with distinctive characteristics that may be advantageous in cochlea gene therapy. Although the general advantages and disadvantages of these vectors had been evaluated, there were no data available on the delivery of extrogene to marginal cells （MCs） of the stria vascularis （SV） in primary culture using nonviral and viral vectors. Methods In this study, the rat cochlear SV MCs were transfected with plasmid vector-pEGFPN1, rAd5, and rAAV2. The transfection efficiency, the incidence of cellular apoptosis and cell＇s activity were evaluated after transfection. Results The transfection rates were as follows： pEGFP-N1, 23.65-45%; rAAV2, 46.89-88%; and rAd5, 78.89-99%. The MCs viabilities after transfection were as follows： PEGFP-N1, 75.79-85.93% （average 80.7%）; rAAV2, 97.54-99.76%（average 98.32%）; rAd5, 88.62-93.3%（average 91.00%）; and normal control, 98.93-99.89%（average 99.27%）. The proportions of early apoptotic cells, end-stage apoptotic and dead cells were as follows： pEGFPN-1-transfected MCs, 39.8%; rAAV2- transfected MCs, 2.4%; and rAd5-transfected MCs, 9.2%. Conclusion Our results indicated that viral vectors were more reliable and efficient vehicles for gene transfer to the SV MCs of cochlea compared to plasmid vector. Given its high transfection efficiency and low cytotoxicity, rAd5 was more suitable for transfection of SV MCs in vitro.

关 键 词：质粒pEGFP—N1 血清5型腺病毒 血清2型腺相关病毒 转染 边缘细胞 大鼠 

分 类 号：R339.16[医药卫生—人体生理学] R394.8[医药卫生—基础医学]