重组腺病毒构建及转染培养耳蜗Corti器和螺旋神经节细胞的实验研究

目的构建携带绿色荧光蛋白（GFP）基因的重组腺病毒,观察GFP基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞的表达。方法通过细菌内同源重组方法构建携带有绿色荧光蛋白基因的重组腺病毒（Ad-GFP）,观察重组腺病毒转染培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,不同时间内绿色荧光蛋白基因的表达情况、表达部位及病毒对培养耳蜗Corti器和螺旋神经节细胞的影响。结果构建的重组腺病毒经酶切电泳鉴定正确;荧光显微镜下观察腺病毒转染体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,12 h即开始表达绿色荧光蛋白基因,24 h达到高峰,表达时间可持续1周;倒置显微镜观察转染后的Corti器和螺旋神经节细胞形态结构及生长无明显改变。结论本实验成功构建了携带绿色荧光蛋白（GFP）基因的重组腺病毒,通过该方法构建的腺病毒可以介导绿色荧光蛋白（GFP）基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞中表达,但腺病毒本身对体外培养的耳蜗Corti器和螺旋神经节细胞生长无明显影响,为通过腺病毒进行内耳基因治疗提供了理论依据。

Construction of recombinant adenovirus and its transgenic expression in cultures of Corti''''s organ and spiral ganglion cells

Objective To construct recombinant adenovirus carrying the gene for green fluorescent protein(Ad-GFP) and observe the expression of GFP in the cultures of Corti's organ and spiral ganglion cells of the neonatal rat.Methods The Ad-GFP was constructed by homologous recombination in bacteria.The expression,distribution of GFP,and the effect of adenovirus on the cultures of Corti's organ and spiral ganglion cells of the neonatal rat were observed at different time points after transfection.Results The constructed recombination was proved to be correct by the agarose gel electrophoresis after restriction digestion.The cultures of Corti's organ and spiral ganglion cells of the neonatal rat began to express GFP gene 12 h after transfection with virus.Gene expression reached a peak level after 24 h and persisted for 1 week.The structure and growth of Corti's organ and spiral ganglion cells were not significantly changed after transfection.Conclusion The recombinant Ad-GFP has been constructed successfully.The adenovirus can mediate GFP expression in the cultures of Corti's organ and spiral ganglion cells of the neonatal rat without great influence on the cultures.Therefore,the constructed recombinant adenovirus may have important value for gene therapy of the inner ear.