腺病毒携带GFP在豚鼠耳蜗基因转导的形态结构与功能改变

目的　带有绿色荧光蛋白 (GFP)基因的腺病毒注入豚鼠耳蜗后 ,观察不同时间段GFP基因的表达和分布情况及手术操作对听力的影响 ,为内耳基因治疗提供理论依据。方法　 1 5只杂色豚鼠术前及术后行听性脑干反应 (ABR)和畸变耳声发射 (DPOAE)检查 ,空白对照组经圆窗或底回钻孔注入人工外淋巴液。实验组注入带有GFP基因的腺病毒。分别于 3、5、7和 1 0d后取材 ,耳蜗标本经硝酸银染色后铺片。结果　腺病毒注入耳蜗后对听阈影响不大。术前术后的DP值有差异。荧光显微镜下可见 3d组表达产物最高。 7d后逐渐减弱 ,1 0d后更弱 ,表达产物主要分布于支持细胞、螺旋神经节细胞和基底膜下的间皮细胞。对照组均无绿色荧光。结论　通过圆窗或底回注入腺病毒对听阈没有影响。单一位点的接种就能使神经营养因子通过耳蜗液扩散到整个耳蜗。有效的基因转移在耳蜗是可行的 ,但表达相对短暂。

Change of hair cell morphology and auditory function in guinea pigs filled with adenoviral mediated GFP

Objective To observe the expression and cellular distribution of GFP gene in the cochlea, and to assess the effect of the operation on hearing. Methods Fifteen mixed coloured guinea pigs were used in this study. The auditory function was assessed by measuring the auditory brainstem response (ABR) and distortion products otoacoustic emission (DPOAE). The experimental and control guinea pigs were inoculated with adenovirus carrying the reported gene GFP, and the control group was inoculated with artificial perilymphatic fluid. On 3 days, 7 days and 10 days the animals were killed and the cochleas were stained with AgNO 3. Results ABR showed that the operation had little significant effect on the ABR thresholds and DPOAE showed there was significant difference between preoperation and postoperation.The gene expression reached a high level three days after the adenovirus administration and reduced thereafter. In the control animals there was no expression.Conclusion The gene inoculated through the round window or basal turn can be spread to all the cochlear turns without causing any effect on the hearing threshold. These results suggest that efficient gene transfer into the inner ear can be achieved, but its expression is transiant.