衔接蛋白AP-2在不同发育时期小鼠耳蜗毛细胞中的表达  

Expression of Adaptin-2 in Different Stages of Mouse Cochlea

作　　者：顾翔[1] 陈知己 蔡婷[1] 宋锐[1] 周小清[1] 袁伟[1] 

机构地区：[1]第三军医大学西南医院耳鼻咽喉头颈外科,重庆400038

出　　处：《听力学及言语疾病杂志》2015年第6期607-611,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金(81470694;30973301;81271080);重庆市自然基金(CSTC2009BB5170)联合资助

摘　　要：目的通过检测不同发育阶段小鼠耳蜗毛细胞衔接蛋白AP-2蛋白的表达,初步探讨其在听觉发生、形成中的作用。方法选用出生后7日(P7)、15日(P15)、35日(P35)及16月龄小鼠各20只,分别代表新生小鼠、听觉发育阶段小鼠、听觉发育成熟小鼠及老年小鼠,对其进行ABR检测,用免疫荧光标记技术结合激光共聚焦显微镜观察各发育阶段小鼠耳蜗AP-2蛋白的定位与表达,用qRT-PCR技术检测小鼠耳蜗AP-2基因mRNA的表达。结果 P15、P35及16月龄小鼠ABR反应阈分别为18.67±1.21、13.83±1.47、37.83±7.68dB nHL,P7组小鼠未引出反应波形。在小鼠内耳发育不同阶段均有不同程度的AP-2蛋白表达,主要定位于耳蜗内毛细胞胞浆,集中表达于内毛细胞基底部与带状突触靠近传入神经元区域,P7、P15、P35及16月龄组小鼠耳蜗AP-2蛋白荧光染色密度值分别为190.91±17.27、494.06±27.63、838.41±38.23和682.65±72.22;AP-2mRNA相对表达量分别为0.53±0.09、1.03±0.02、1.00±0.09和1.03±0.06;与新生小鼠相比较,随小鼠听觉的产生和形成,耳蜗AP-2蛋白表达水平逐渐升高(P<0.01),免疫荧光染色提示老年性聋小鼠AP-2蛋白在内毛细胞的表达量较P35小鼠减少(P<0.05),但qRT-PCR分析AP-2基因mRNA的表达水平与成熟小鼠(P15)差异无统计学意义(P>0.05)。结论在小鼠听觉发育成熟阶段,随小鼠日龄增加耳蜗AP-2蛋白表达逐渐增强,提示AP-2蛋白的表达水平可能与听觉的发生、形成和维持密切相关。Objective To investigate the expression of adaptin-2（AP-2）in different stages of mouse cochlea and its probable role in the auditory generation and formation.Methods Mice were divided into 4experimental groups by age（7days old,15 days old,35 days old,16 months old）,which respectively represented the newborn mice,developmental mice,mature mice and old mice.Auditory brainstem response（ABR）,laser scanning confocal microscopy（LSCM）,immune-fluroscence histochemistry and qRT- PCR were used in this study.Results For the 15 days old,35 days old,16 months old groups,ABR average threshold was 18.67±1.21 dB nHL,13.83±1.47 dB nHL,37.83±7.68 dB nHL,respectively,for the 7days old groups no responses were observed.The AP-2immunoreactivity was found in all the stages of mice cochlea inner hair cell（IHC）cytoplasm,especially in IHC basal part,nearby the ribbon synapse.For the 7days old,15 days old,35 days old,16 months old groups,immune-fluroscence histochemistry IMV（intensity mean value）were 190.91±17.27,494.06±27.63,838.41±38.23,682.65±72.22,respectively.For the 7days old,15 days old,35 days old,16 months old groups,mRNA RQ（relative quantity）were 0.53±0.09,1.03±0.02,1.00±0.09,1.03±0.06,respectively.Developmental mice expressed significantly higher than those of the newborn in the AP-2protein expression level which was measured by immuno-fluorescence histochemistry and qRT PCR（P〈0.01）.There was no significant difference between old and mature mice in the AP-2protein expression level measured by qRT PCR（P〉0.05）,but mature mice had significant advantages by immuno-fluorescence histochemistry.Conclusion AP-2protein expression level may closely related to the auditory formation and maintenance because its expression gradually increases with age in mice.

关 键 词：衔接蛋白 内毛细胞 年龄相关性听力损失 小鼠 

分 类 号：R764.456[医药卫生—耳鼻咽喉科]