新生大鼠耳蜗Klliker器在体凋亡的研究  

The Apoptosis of the Klliker Organ in the Cochlea of Newborn Rat in Vivo

作　　者：杨军[1] 何圆圆[1,2] 

机构地区：[1]上海交通大学医学院附属新华医院耳鼻咽喉-头颈外科上海市耳鼻疾病转化医学重点实验室上海交通大学医学院耳科学研究所,上海200092 [2]南京医科大学附属无锡人民医院耳鼻咽喉科

出　　处：《听力学及言语疾病杂志》2016年第4期371-376,共6页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金(81170919,81470689);国家科技部973课题(2014CB541705);上海市科委重大项目(14DJ1400201,14DZ2260300)资助

摘　　要：目的了解不同天龄新生大鼠耳蜗Klliker器的形态变化,研究凋亡相关因子的mRNA及蛋白的表达水平,探讨Klliker器在听觉功能发育过程中凋亡的机制。方法选取出生后不同天龄的Sprague-Dawley大鼠共192只,其中出生后1天(P1)、5天(P5)、12天(P12)各6只大鼠耳蜗冰冻切片后,通过苏木精-伊红染色及免疫荧光染色等方法,观察耳蜗Klliker器的形态结构变化;取P1大鼠6只行耳蜗基底膜免疫荧光观察;提取出生后1、3、5、7、10、12及14天(P1、P3、P5、P7、P10、P12和P14)大鼠耳蜗基底膜mRNA(各6只)及蛋白(各18只),运用real-time PCR及蛋白质印迹的方法,观察出生后各天龄组大鼠耳蜗基底膜Klliker器凋亡过程中bcl-2、caspase-3、caspase-8及caspase-9的表达规律。结果出生后大鼠听力出现之前其耳蜗Klliker器支持细胞的形态自顶回向底回从高柱状向矮柱状变化,同时支持细胞的数量逐渐减少。出生后不同天龄大鼠耳蜗基底膜的caspase-3、caspase-8、caspase-9及bcl-2的mRNA和蛋白的表达水平均呈明显的时间依赖性。结论在大鼠出生后、听力出现前耳蜗发育的整个阶段,Klliker器自底回向顶回逐渐发生退化;caspase-3、caspase-8、caspase-9及bcl-2的mRNA和蛋白的表达表现为时间依赖性。Objective To explore the apoptosis mechanisms of the Klliker organ during the development of the auditory function,through the obser vation of the morphological changes,the expression of the apoptosis-related mRNA or protein of the Klliker organ in the cochlea of newborn rats.Methods A total of 192 SpragueDawley rats at different days after parturition（P）were used.There were P1,P5,and P12rats（6rats in each group）for hematoxylin-eosin staining and immunohistochemistry to investigate the morphological changes using frozen sections of the cochlea.Six rats at P1 were used for the immunofluorescence of the cochlear basal membrane.The sensory epitheliums in P1,P3,P5,P7,P10,P12 and P14rats（6rats each）were separated to extract mRNA（6rats each）and protein（18rats each）,respectively.The differential expressions of the bcl-2,caspase-3,caspase-8and caspase-9through the real-time PCR and western blot during the apoptotic process of the Klliker organ were studied.Results After birth,the supporting cells in the Klliker organ showed the morphological change from the high columnar to short columnar and the disappearance with a base-to-apex gradient.The results of real-time PCR and western blot showed the expression pattern of bcl-2,caspase-3,caspase-8and caspase-9mRNA and protein in the cochlear sensory epithelium at 1st,3rd,5th,7th,10 th,12th and 14 th day.This indicates a significant time-dependent along the course of development.Conclusion After birth,but prior to the onset of hearing,the Klliker organ in the cochlea of newborn rats degraded with a base-to-apex gradient throughout the developmental period of the cochlea.The expression of the caspase-3,caspase-8,caspase-9and bcl-2in the Klliker organ during this period also showed in a time-dependence pattern.

关 键 词：大鼠 耳蜗 K0lliker器 支持细胞 凋亡 

分 类 号：R339.16[医药卫生—人体生理学]