CAJ | 학술논문

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±7．68 dB 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A P －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 －2 mRNA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。
목적：통과검측불동발육계단소서이와모세포함접단백AP －2단백적표체，초보탐토기재은각발생、형성중적작용。방법선용출생후7일（P7）、15일（P15）、35일（P35）급16월령소서각20지，분별대표신생소서、은각발육계단소서、은각발육성숙소서급노년소서，대기진행ABR검측，용면역형광표기기술결합격광공취초현미경관찰각발육계단소서이와AP－2단백적정위여표체，용qRT －PCR기술검측소서이와AP－2기인mRNA적표체。결과 P15、P35급16월령소서ABR반응역분별위18．67±1．21、13．83±1．47、37．83±7．68 dB nHL ，P7조소서미인출반응파형。재소서내이발육불동계단균유불동정도적AP－2단백표체，주요정위우이와내모세포포장，집중표체우내모세포기저부여대상돌촉고근전입신경원구역，P7、P15、P35급16월령조소서이와A P －2단백형광염색밀도치분별위190．91±17．27、494．06±27．63、838．41±38．23화682．65±72．22；AP －2 mRNA상대표체량분별위0．53±0．09、1．03±0．02、1．00±0．09화1．03±0．06；여신생소서상비교，수소서은각적산생화형성，이와AP－2단백표체수평축점승고（P＜0．01），면역형광염색제시노년성롱소서AP－2단백재내모세포적표체량교P35소서감소（P＜0．05），단qRT －PCR분석AP－2기인mRNA적표체수평여성숙소서（P15）차이무통계학의의（ P＞0．05）。결론재소서은각발육성숙계단，수소서일령증가이와AP－2단백표체축점증강，제시AP－2단백적표체수평가능여은각적발생、형성화유지밀절상관。
Objective To investigate the expression of adaptin-2(AP-2) in different stages of mouse coch‐lea and its probable role in the auditory generation and formation .Methods Mice were divided into 4 experimental groups by age (7 days 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 7 days old groups no responses were observed .The AP-2 im‐munoreactivity 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 7 days old ,15 days old ,35 days old ,16 months old groups ,immune-flu‐roscence 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 7 days 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 -2 protein expression level which was measured by immuno -fluores‐cence histochemistry and qRT PCR(P<0 .01) .There was no significant difference between old and mature mice in the AP-2 protein expression level measured by qRT PCR (P> 0 .05) ,but mature mice had significant advantages by immuno-fluorescence histochemistry .Conclusion AP-2 protein expression level may closely related to the au‐ditory formation and maintenance because its expression gradually increases with age in mice .