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目的:构建一个神经营养因子3(NT3)和绿色荧光蛋白(EGFP)基因共表达重组腺病毒载体,确定Ad/NT-3对离体耳蜗的转染效率和NT3对螺旋神经元存活的作用。方法:使用pAdeasy-1和pAdTrack CMV腺病毒载体系统,产生带绿色荧光标记的NT3重组腺病毒,转染培养新生的大鼠耳蜗基底膜,观察病毒的转染效率。对培养15d的耳蜗进行神经丝蛋白200免疫荧光染色,计数螺旋神经元,观察NT3对神经存活的影响。结果:重组NT3病毒能够转染整个耳蜗组织中的各型细胞。以外沟细胞最高,约为49%,其次为齿间细胞,为27%。仅有少量的毛细胞和螺旋神经元被转染。Ad/NT-3重组腺病毒处理15d的耳蜗螺旋神经元存活的数目多于Ad/EGFP腺病毒转染的耳蜗。结论:构建的Ad/NT-3重组腺病毒能够同时在耳蜗组织中表达EGFP和NT3蛋白,主要表达于外沟细胞和齿间细胞,这些细胞释放NT3能够维持耳蜗神经元存活。
OBJECTIVE: To construct a recombinant adenovirus vector containing NT3 and EGFP gene to determine the transfection efficiency of Ad / NT-3 on cochlear implants and the effect of NT3 on the survival of spiral ganglion neurons. Methods: The adenovirus vector system of pAdeasy-1 and pAdTrack CMV was used to generate the recombinant adenovirus with green fluorescence-labeled NT3. The recombinant adenovirus was transfected into neonatal rat cochlea to observe the transfection efficiency. Neurofilament 200 immunofluorescent staining was performed on cochlea cultured for 15 days. Spiral neurons were counted to observe the effect of NT3 on neural survival. Results: The recombinant NT3 virus can transfect various types of cells in the entire cochlear tissue. Outside the highest groove cells, about 49%, followed by interdental cells, 27%. Only a few hair cells and spiral neurons are transfected. The number of surviving cochlear spiral neurons treated with Ad / NT-3 recombinant adenovirus for 15 days was higher than that of Ad / EGFP adenovirus transfected cochlea. CONCLUSION: The constructed Ad / NT-3 recombinant adenovirus can express both EGFP and NT3 protein in cochlear tissue at the same time, mainly expressed in outer groove cells and interdental cells. The release of NT3 can maintain the survival of cochlear neurons.