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

邓志宏;王锦玲;邱建华;刘顺利;王成济;杨安钢

1:第四军医大学西京医院耳鼻咽喉科

2:第四军医大学西京医院耳鼻咽喉科

3:第四军医大学西京医院耳鼻咽喉科

4:第四军医大学西京医院耳鼻咽喉科

摘要
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 - 3能够在较长时间内持续表达
关键词
神经营养素-3;基因克隆;真核表达载体;脂质体;耳蜗
基金项目(Foundation):
作者
邓志宏;王锦玲;邱建华;刘顺利;王成济;杨安钢
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