阳离子脂质体介导NT-3基因转染豚鼠耳蜗的实验研究

Cationic Liposome Medicated Transgene Expression in Guinea Pig Cochlea

目的 探讨阳离子脂质体介导的NT - 3基因转染对豚鼠耳蜗形态及功能的影响。方法 克隆人NT- 3cDNA基因 ,构建携带绿色荧光蛋白 (EGFP)报告基因的重组质粒pIRES2 -EGFP -NT3。采用脂质体介导的方法 ,将重组质粒转染豚鼠耳蜗 ,分别于术后 1天、2周、4周观察转染基因在耳蜗及脑干等部位的表达和分布情况。同时进行耳声发射及ABR测试 ,了解基因转染对豚鼠耳蜗听功能的影响。结果 成功克隆人NT - 3cDNA基因并构建重组质粒pIRES2 -EGFP -NT3。重组质粒转染豚鼠耳蜗后 ,荧光显微镜下观察发现耳蜗螺旋神经节、神经纤维、Corti器、血管纹等均可见绿色荧光 ,但 4周时荧光蛋白的表达强度较 1天时明显减弱。而CY3标记的NT - 3免疫荧光染色结果基本与EGFP的表达情况一致。此外 ,对侧耳蜗及第四脑室脉络膜处亦可见较强的绿色荧光。耳蜗基底膜铺片见内外毛细胞结构完整。转染前后豚鼠耳声发射及ABR阈值均无明显改变。结论 阳离子脂质体介导的NT - 3基因转染对豚鼠耳蜗形态及功能无明显影响 ;外源性NT -

Objective To study the expression of cationic liposome medicated transgene expression in the guinea pig cochlea.Methods The NT-3 cDNA gene was cloned and its eukaryotic expression vector(pIRES2-EGFP-NT3) with the reporter gene-EGFP was constructed.Then the liposome-plasmid complex was injected into the guinea pig cochlea,and the expression of the reporter gene-EGFP and NT-3 were observed respectively after 1 d?2 W and 4 W while the audition of the guinea pig were measured.Results The human NT3-cDNA was successfully cloned and its eukaryotic expression vector(pIRES2-EGFP-NT3) was constructed.Transgene expression of the guinea pig injected with the plasmid-cationic liposome complex was persistent up to 4ws.The transfected tissues included the spiral ganglia,the never fibre,the organ of Corti,and the stria vascularis.Transgene expression was detected in the ipsilateral cochlea,but also the contralateral cochlea,even the brain tissues.The hair cells with the FITC-phalloidin immunohistochemical dyeing showed no hurt,even with the test of DPOAE and ABR.Conclusion This study represents the successful use of cationic liposomes for cochlear gene transfer,thus providing a safe,rapid and effective way in gene therapy for inner ear disorders.