CAJ | 학술논문

目的研究GJB2基因条件敲除（cCx26Pax2Cre）小鼠耳蜗DR5表达情况，探讨GJB2基因突变导致耳聋的机制。方法 cCx26Pax2Cre小鼠为实验组，BALB/C小鼠为对照组，分别取P8、P12和P21时小鼠耳蜗行冰冻切片，利用荧光标记免疫组织化学方法和激光共聚焦显微镜技术观察死亡受体5（death receptor，DR5）蛋白在耳蜗的表达情况，用Image Pro Plus 6.0计算平均光密度值，SPSS 18.0进行统计分析。结果 P8时DR5主要表达在柯蒂氏器外侧的支持细胞、耳蜗外侧壁Ⅱ型纤维细胞、盖膜，随着日龄的增加，表达逐渐向内侧延伸到内侧支持细胞，P12和P21时还出现了螺旋缘、毛细胞、螺旋神经节细胞、螺旋凸DR5表达阳性，而且表达强度随日龄增加而逐渐变强。在野生小鼠DR5也有表达但较弱。与野生型小鼠相比， cCx26Pax2Cre小鼠耳蜗蜗轴螺旋管内神经纤维DR5表达的光密度值明显增大，经统计学分析两者具有显著性差异（P<0.05）。结论 cCx26Pax2Cre小鼠缝隙连接功能异常，可能导致耳蜗细胞出现葡萄糖短缺，导致三磷酸腺苷(adenosine-triphosphate， ATP)不足，引起DR5的表达上调，直接激活凋亡的死亡受体途径，或间接激活线粒体通路使凋亡信号进一步放大，导致耳蜗细胞凋亡的发生，最终引起cCx26Pax2Cre小鼠听力下降。
목적연구GJB2기인조건고제（cCx26Pax2Cre）소서이와DR5표체정황，탐토GJB2기인돌변도치이롱적궤제。방법 cCx26Pax2Cre소서위실험조，BALB/C소서위대조조，분별취P8、P12화P21시소서이와행빙동절편，이용형광표기면역조직화학방법화격광공취초현미경기술관찰사망수체5（death receptor，DR5）단백재이와적표체정황，용Image Pro Plus 6.0계산평균광밀도치，SPSS 18.0진행통계분석。결과 P8시DR5주요표체재가체씨기외측적지지세포、이와외측벽Ⅱ형섬유세포、개막，수착일령적증가，표체축점향내측연신도내측지지세포，P12화P21시환출현료라선연、모세포、라선신경절세포、라선철DR5표체양성，이차표체강도수일령증가이축점변강。재야생소서DR5야유표체단교약。여야생형소서상비， cCx26Pax2Cre소서이와와축라선관내신경섬유DR5표체적광밀도치명현증대，경통계학분석량자구유현저성차이（P<0.05）。결론 cCx26Pax2Cre소서봉극련접공능이상，가능도치이와세포출현포도당단결，도치삼린산선감(adenosine-triphosphate， ATP)불족，인기DR5적표체상조，직접격활조망적사망수체도경，혹간접격활선립체통로사조망신호진일보방대，도치이와세포조망적발생，최종인기cCx26Pax2Cre소서은력하강。
Objective To observe the expression of death receptor 5 (DR5) in the cochleae of GJB2 conditional knockout mice (cCx26Pax2Cre) and to explore the mechanism of deafness caused by GJB2 gene mutations. MethodsThe frozen section technique was used to process the cochleae of cCx26Pax2Cre mice (experimental group) and BALB / C mice (control group) at P8, P12 and P21. The immunofluorescence technique was used to observe the expression of DR5 in the cochleae and the Image Pro Plus was applied to analyze the average optical density. The results were analyzed with SPSS 18.0 software.Results At the stage of P8, DR5 mainly expressed in the outer supporting cells of the organ of Corti, tectorail membrane, type Ⅱ fibrocytes in spiral ligament. At the stage of P12 and P21,the expression gradually extended to the inner supporting cells, spiral limbus, hair cells, spiral ganglion cells and spiral prominence. The intensity of expression increased with the increasing age. DR5 also expressed in wild-type mice, but the intensity was lower than that in cCx26Pax2Cre mice at the same stage. Compared with the wild-type mice, the expression of DR5 in nerve fiber in spiral tube of cCx26Pax2Cre mice was much stronger. The difference of optical density values showed statistical significance(P<0.05).Conclusion It is speculated that after the GJB2 gene conditional knockout, the cochlear energy-intensive cells are short of glucose and ATP and DR5 overexpression ensues. The overexpression of DR5 triggers the death receptor pathway causing apoptosis directly or activates the mitochondria pathway indirectly which amplifies the apoptotic signals. The final result of the above activated pathways is the apoptosis in a wide range of cochlear cells and hearing loss in cCx26Pax2Cre mice.