腺相关病毒介导的GDNF对卡那霉素致耳蜗损害的保护性研究  

Effects of adeno-associated virus vector-mediated GDNF gene transfer on kanamycin-induced cochlear toxicity

作　　者：刘玉和[1] 王成元[2] 柯肖枚[1] 肖水芳[1] 

机构地区：[1]北京大学第一医院耳鼻咽喉头颈外科,北京100034 [2]卫生部中日友好医院耳鼻咽喉头颈外科,北京100029

出　　处：《中国听力语言康复科学杂志》2006年第6期14-18,共5页Chinese Scientific Journal of Hearing and Speech Rehabilitation

摘　　要：目的利用腺相关病毒(adeno-associatedvirus,AAV),不同血清型对不同宿主细胞转导率存在差异的特点,探讨不同血清型AAV对耳蜗各种细胞尤其是毛细胞的转染率,进一步探讨高转染率的血清型AAV转导神经胶质细胞源性神经营养因子(glialcellline-derivedneurotrophicfactor,GDNF)对卡那霉素致鼠耳蜗损害的保护作用。方法采用不同血清型的AAV携载标记基因绿荧光蛋白EGFP,微注射到鼠耳蜗,荧光显微镜检测耳蜗内EGFP的表达分布和强度。应用筛选出的高转导率的血清型AAV构建携载GDNF的载体,微注射到卡那霉素耳聋模型鼠耳蜗内,经脑干诱发电位检测听力阈值,ELISA法检测耳蜗外淋巴液GDNF蛋白表达,免疫组化检测GDNF蛋白在细胞内的表达,耳蜗毛细胞和螺旋神经节细胞染色检测细胞的凋亡。结果荧光显微镜结果显示七种AAV血清型转导的EGFP均可分别在耳蜗的不同细胞内表达,其中AAV3-EGFP特异性在耳蜗内毛细胞表达,AAV1-EGFP在耳蜗内毛细胞等高效表达。在卡那霉素耳聋模型鼠中,注射AAV1-GDNF的耳蜗听力阈值比对侧耳低,GDNF在注射耳蜗内的表达水平比对照组高,其在耳蜗内的表达分布与EGFP的表达相似,治疗耳的耳蜗毛细胞和螺旋神经节细胞比对照耳幸存多。结论作为耳蜗基因治疗的载体,AAV血清型具有高转导率。AAV1型载体转导的GDNF保护了耳蜗功能,可以作为一个有价值的方法预防氨基糖甙类抗生素导致的耳聋。本研究为今后耳蜗毛细胞的进一步研究,以及对由于单基因突变导致的隐性遗传性耳聋开展耳蜗的基因治疗提供依据。Objective To evaluate the transduction efficiency of the serotypes of adeno-associated virus （AAV） as vector for cochlear gene therapy, the otoprotective efficacy of AAV-mediated glial cell line-derived neurotrophic factor （GDNF） gene on aminoglycoside-induced ototoxicity. Methods Seven serotypes of AAV （AAVl-5, 7, 8） harboring the enhanced green fluorescent protein （EGFP）, and AAVl vector encoding the mouse GDNF, were microinjected into the cochlea. The expression pattern of AAV-mediated EGFP was determined with fluorescence microscope and screening. The cochlear function was analyzed with evoked auditory brainstem responses （ABR）. The GDNF expression was detected with ELISA and histochemistry, and the cochlear ganglion cells and hair cells were checked for evaluation. Results Each of these serotypes successfully targetted distinct cell types within the cochlea. In contrast to other serotypes, AAV3 vector specifically and most efficiently transduced the cochlear inner hair cells in vivo, while AAVl could infect inner hair cells as well as other cell types. GDNF level in the cochlear inoculated with AAV1-GDNF was higher than that in the controls, and GDNF expression pattern in the cochlea was similar to AAV1-mediated EGFP expression. The ABR monitoring revealed that the inoculated cochlea with AAV1-GDNF vector showed better ABR thresholds after exposure to the ototoxins, as compared to the contralateral side. Fewer missing spiral ganglion cells and hair cells were found in the inoculated cochlear, as compared with the controls. Conclusion The results demonstrated the feasibility of gene therapy for cochlear application, and AAV1-mediated GDNF expression preserved the cochlear function from the aminoglycosides-induced ototoxicity, suggested that AAV1-mediated expression of GDNF could be developed as a valuable prevention against aminoglycoside-induced damage.

关 键 词：腺相关病毒 血清型 耳聋 卡那霉素 基因转染/基因治疗 

分 类 号：R764[医药卫生—耳鼻咽喉科]