Objective To test the mechanism and upstream pathway of outer hair cell apoptosis in Cadherin 23(Cdh23)gene mutant mice.Method The mutant Cdh23erl/erl(erl)mice were collected as the study group,while the C57BL/6J(B6)mice were chosen as the control group.A total of 70 mice per group were used in this study.The study group and control group underwent auditory-evoked brainstem response (ABR)tests at the same age.The terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) assay was performed to detect outer hair cell(OHC)apoptosis.The qRT-PCR was conducted to test the expression of ER stress markers immunoglobulin-binding protein(BiP)and C/EBP homologous protein (CHOP)mRNA.The expression and location of BiP and CHOP protein in OHC were detected by immunostaining.The expression of BiP protein in cochleae was identified by Western blot.The expression and location of CDH23 protein in OHC were discovered by immunostaining.Results The ABR thresholds in erl mice were significantly higher than those in B 6 mice at the age of 1 and 3 months(both P<0.05). The surface preparation with TUNEL staining confirmed OHC apoptosis in erl mouse cochleae which showed a higher TUNEL positive cell ratio than B6 mouse(t=11.291,P<0.01).The ER stress marker Bip and Chop mRNA were upregulated in the erl mouse inner ear,when compared with those in the B6 mouse(both P<0.05).The BiP protein extracted from the erl mouse cochleae was significantly higher than that of B 6 mouse measured by Western blot(t=3.66,P=0.02).Immunostaining showed that BiP and CHOP were highly detected in the OHC in erl mouse cochleae, and was mainly detected in the perinuclear region of OHC. However,a bare BiP and CHOP signal were shown in B6 mouse cochleae.The CDH23 protein was specifically localized at the top of the OHC in B6 mice, indicating the localization of the tip links in hair bundle stereocilia.On the contrary,the CDH23erl protein was found to be localized from the top to the nuclei of the OHC in erl mice.Portions of the CDH23erl proteins failed to reach the top of the hair bundles and remained in the OHC cytoplasm.Conclusion As the downstream response of the Cdh23 gene mutation, portions of the mutant CDH23erl protein was accumulated in ER lumen resulting in the increase of ER loading and ultimately triggered ER stress and hair cell apoptosis in erl mouse cochleae.%目的 探讨内质网应激参与钙黏蛋白23(Cadherin 23,Cdh23)基因突变小鼠耳蜗外毛细胞损伤的机制.方法 采用纯合型Cdh23基因突变小鼠(erl小鼠)为实验组,C57BL/6J小鼠(B6)为对照组,每组各70只.测试两组小鼠ABR阈值,脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)技术检测细胞凋亡.通过实时荧光定量PCR、蛋白免疫印迹(Western blot)及免疫荧光染色,检测内质网应激标志物免疫球蛋白结合蛋白(immunoglobulin-binding protein, BiP)和核转录因子C/EBP同源蛋白(C/EBP homologous protein,CHOP)在小鼠耳蜗中的表达及分布,观察CDH23蛋白在耳蜗外毛细胞的定位.结果 1月龄及3月龄erl小鼠8、16、32 kHz ABR阈值均显著高于对照组B6小鼠,差异具有统计学意义(P值均<0.05).erl小鼠耳蜗外毛细胞TUNEL阳性细胞率显著高于B6小鼠,差异具有统计学意义(t=11.291,P<0.01).Bip及Chop mRNA在erl小鼠耳蜗表达明显高于B6小鼠,BiP蛋白在erl小鼠耳蜗表达明显高于B6小鼠,差异具有统计学意义(P值均<0.05).B6小鼠耳蜗外毛细胞未检测到BiP及CHOP信号,而erl小鼠耳蜗外毛细胞细胞质中BiP及CHOP特异性高表达.CDH23蛋白在B6小鼠外毛细胞远离细胞核的顶端表达;在erl小鼠的耳蜗外毛细胞,突变型CDH23erl蛋白部分表达于远离细胞核的顶端,部分滞留于细胞质.结论 小鼠Cdh23基因突变导致CDH23蛋白质翻译异常,生成突变型CDH23erl蛋白,部分突变型蛋白转运障碍,滞留于内质网腔,引发内质网应激并最终导致外毛细胞损伤.
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