C57BL/6J小鼠耳蜗内毛细胞传入神经突触的免疫学标记观察及其意义  

Immunostaining of cochlear ribbon synapse in adult C57BL/6J mice

作　　者：施磊[1] 赵宁[1] 陈丽平[1] 商秀丽[1] 徐义策[2] 王雪峰[2] 柳柯[1,3] 

机构地区：[1]中国医科大学附属第一医院耳鼻咽喉-头颈外科,沈阳110001 [2]辽宁医学院附属第一医院耳鼻咽喉-头颈外科,锦州121001 [3]中国人民解放军总医院耳鼻咽喉-头颈外科,中国人民解放军总医院耳鼻咽喉研究所,北京100853

出　　处：《中华耳科学杂志》2010年第4期456-460,共5页Chinese Journal of Otology

基　　金：国家自然基金项目(31040038);中国博士后科学基金(20100470103,201003779)资助项目

摘　　要：目的探讨小鼠耳蜗内毛细胞传入神经突触的免疫学标记特点及其应用方法。方法在成年的C57BL/6J小鼠上剥取新鲜的耳蜗基底膜标本,灌洗及漂洗处理之后分别使用Ctbp2(C-terminal binding protein 2,C末端结合蛋白2;1∶200),GluR 2/3(glutamate receptor2/3,AMPA receptor subunit 2/3,谷氨酸受体亚单位2/3;1∶200)和Dapi(4',6-Diamidino-2-phenylindole,4',6-二脒基-2-苯基吲哚)染色剂对内毛细胞突触的前膜特异结构RIBEYE、突触后膜的谷氨酸受体以及细胞核进行单独、双重和三重免疫标记,并利用激光共聚焦显微镜能够连续扫描和叠加图像的特性,观察耳蜗内毛细胞带状突触的表达、空间分布情况以及与内、外毛细胞之间的位置关系。结果单独应用Ctbp2可以清晰显示内毛细胞突触前膜所标记的结构,结合使用抗体GluR2/3进行免疫双标则可以完整显示内毛细胞突触前后膜的相关蛋白,而结合应用Dapi细胞核染色的方法可以明确标记其存在的位置以及和内、外毛细胞之间的关系,如果在此基础上加上激光共聚焦显微镜下的平光图像,则会使标记结果更加清晰、客观和准确。结论本方法可以准确显示耳蜗内毛细胞带状突触的表达、空间分布情况以及与内外毛细胞之间的位置关系,如果在实验中合理应用多重免疫荧光标记方法,将会对不同生理和病理条件下深入研究耳蜗内毛细胞突触的可塑性及其在听力变化中的作用具有重要的价值。Objective To study characteristics of immunolabling of cochlear ribbon synapses in adult C57BL/6J mice and its significance in research.Methods Fresh apical coil organ of Corti specimens were obtained for whole mount staining with Ctbp2 or GluR2/3 alone,or in various combinations including Ctbp2 with Dapi,GluR2/3 with Dapi,Ctbp2 with GluR2/3 and Ctbp2 with GluR2/3 plus Dapi.Cochlear ribbon synapses were examined using a confocal microscope for patterns of immunostaining.Results Ctbp2 stainging highlighted spots where cochlear ribbon synapses were located and anatomical relations were shown by Ctbp2 and Dapi double staining.When Ctbp2 was combined with GluR2/3,the entire cochlear ribbon synapse was demonstrated,with improved illustration of anatomical connections between ribbon synapses and hair cells in the cochlea when Dapi was added.Conclusion This study demonstrates a useful method for studying ribbon synapse of cochlear inner hair cells,which may benefit future studies.

关 键 词：内毛细胞传入神经突触 RIBEYE/Ctbp2 免疫标记 激光共聚焦显微镜 

分 类 号：R322.923[医药卫生—人体解剖和组织胚胎学] R338.13[医药卫生—基础医学]