不同基因转染方法对小鼠内耳毛细胞转染效率的比较

The Comparative Study of Different Methods for Gene Transfer to Cochlear Hair Cells

目的对比不同的基因转染方法对小鼠内耳毛细胞的转染效率,以探寻简便、高效转染耳蜗毛细胞的方法。方法分离111只新生昆明小鼠(P2)耳蜗螺旋韧带及感染上皮,分别采用电穿孔法介导质粒载体(pGPHI/GFP/Neo)、腺病毒载体(Ad5/CMV/GFP)和慢病毒载体(LV/CMV/GFP)3种方法转染小鼠内耳毛细胞,于转染48小时后在荧光显微镜下观察并比较三种方法基因转染后小鼠内耳毛细胞绿色荧光蛋白(green fluorescence protein,GFP)的表达。结果电穿孔法介导质粒载体转染组和慢病毒载体转染组GFP阳性细胞极少;腺病毒载体转染组效率最高,耳蜗中回外毛细胞GFP阳性率为90.0%±4.1%,内毛细胞GFP阳性率为5%±0.4%。结论腺病毒载体能更有效地将外源基因导入耳蜗毛细胞内表达,是基因转染耳蜗毛细胞的理想载体。

Objective To obtain an easy and high efficient method for gene transfer to cochlear hair cells ,by comparing three mediating green fluorescent protein （GFP） methods （electroporation ,adenovector and lentivirus vector） .Methods Cochlear sensory epithelium was dissected from anaesthetized P2 mice .Sensory epithelia were transferred onto poly -L -lysine treated cover slides and cultured overnight .Gene transfer was performed by elec‐troporation in medium containing pGPHI/GFP/Neo plasmid or by incubation with diluted recombined adenovirus/lentivirus vector .After 48 hours ,green fluorescence was checked under fluorescence microscope .To confirm the ef‐ficiency of exogenous gene transfer ,real-time PCR was performed using specific primers .Results The transfec‐tion efficiencies of electroporation and lentivirus vector mediated gene transfer were very low .Both immunofluores‐cence and real - time PCR results showed that the transfection efficiency of adenovirus mediated GFP and Bmi1 transfer were relatively higher .The proportion of GFP positive cells in outer hair cells and inner hair cells of middle turn were 90 .0% ± 4 .1% and 5% ± 0 .4% ,respectively .Conclusion Adenovector is more efficient for exogenous gene transfer to cochlear hair cells ,thus adenovector is a good carrier for gene transfer to cochlear hair cells .