大鼠胚胎神经干细胞经蜗窗移植到正常耳蜗的研究

Experimental study on embryonic neural stem cells transplantation into natural rat cochlea via round window

目的探讨感染携带绿色荧光蛋白（green fluorescent protein，GFP）报告基因的重组腺病毒（recombinant adenovirus with GFP，Ad-GFP））的大鼠胚胎神经干细胞（neural stem cell，NSC）经蜗窗径路移植到正常大鼠耳蜗后的存活、分布及报告基因的表达情况，并检测移植后大鼠听力和耳蜗形态结构的变化。方法大鼠随机分为三组：组Ⅰ（感染Ad—GFP的NSC移植组）10只，组Ⅱ（人工外淋巴注射组）10只，组Ⅲ（空白对照组）10只。组Ⅰ及组Ⅱ的手术径路均为经蜗窗途径；两组移植前后分别测试大鼠听性脑干反应（ABR），了解其听力的改变；移植后14d在荧光显微镜下观察耳蜗中轴切片中感染Ad-GFP的NSC的存活、分布及报告基因的表达情况，并通过耳蜗切片HE染色及基底膜铺片硝酸银染色观察耳蜗形态结构及毛细胞数目的变化。结果组Ⅰ及组Ⅱ术后ABR反应阈分别为（33．5±3．4）dB和（32．5±3．5）dB，与移植前相比差异均无统计学意义（P值均〉0．05），两组术后ABR反应阈间的差异亦无统计学意义（t=0．526，P〉0．05）。两组大鼠术后耳蜗形态结构均正常。组ⅠNSC移植入耳蜗14d后，存活的细胞主要分布在耳蜗底回的前庭阶和鼓阶，耳蜗其他各回的前庭阶和鼓阶中也有存活，同时表达强烈的绿色荧光。三组均出现耳蜗外毛细胞的散在缺失，总缺失率均未超过1％，且三组各回的缺失率及总缺失率之间的差异均无统计学意义（P值均〉0．05）；三组耳蜗各回均未见内毛细胞的缺失。结论感染Ad-GFP的NSC经蜗窗径路移植到正常大鼠耳蜗后可以存活并表达GFP基因，移植对大鼠听反应阈及耳蜗形态结构无明显影响。

Objective To investigate the survival of neural stem cell （NSC） infected by recombinant adenovirus with GFP （Ad-GFP）and the expression of GFP in normal rat cochlea and their potential effect on auditory function and cochlea structures via round window transplantation. Methods In comparison with the normal rats without any transplantation （Group Ⅲ ）, normal rat cochleae were transplanted with NSC infected with Ad-GFP （ Group Ⅰ ） or the artificial perilymph （ Group Ⅱ ） via round windows. Auditory functions were monitored by thresholds of auditory brain stem responses （ABR） ; the cochlea structures were examined by HE staining; survivals of implanted NSC were determined by the expression of GFP; survivals of hair cells were accessed by whole mount preparation. Results Neither at pre-transplantation nor at post-transplantation, there were significant differences in the click-ABR thresholds in rats between Group Ⅰ and Group Ⅱ （P 〉 0. 05）. There were no significant differences in these values before and after transplantation in the same rats from each group. After transplantation, the cochlea structures were normal in both Group Ⅰ ＆ Group Ⅱ. Grafted NSC was visualized by the GFP expression in every turn of the cochlea in all animals of Group Ⅰ . There were no significant differences in the loss of outer hair cell （OHC） among three groups. The inner hair cell （IHC） and most OHC were normal in every turns of cochleae of all groups. Conclusions The embryonic NSC infected with Ad-GFP could survive and express the GFP gene in normal rat cochlea after transplantation via round window, which had not obvious affection to auditory function and inner ear pathology of rat cochlea.