细胞色素C在老年SD大鼠耳蜗不同阶段凋亡坏死毛细胞中表达的变化

目的：观察细胞色素c在老年SD大鼠不同阶段凋亡、坏死毛细胞中表达的变化，探讨老年大鼠耳蜗毛细胞死亡的机制。方法：16只SD大鼠分组：青年组8只，2～3月龄；老年组8只，24～26月龄。应用电位反应测听仪检测不同频率短纯音（4、8、16、32和40kHz）诱发的青年组和老年组大鼠双侧ABR。听觉功能测试后，解剖取出双耳听泡，分离耳蜗基膜。分别用细胞核DNA染料碘化丙锭染色不同月龄大鼠耳蜗基膜细胞核，细胞色素C免疫荧光染料标记大鼠耳蜗基膜细胞的细胞色素c蛋白，荧光显微镜下观察不同月龄大鼠耳蜗基膜细胞荧光染色的变化。结果：老年组大鼠的不同频率ABR阈值均高于青年组（P〈0．01）。微弱拘细胞色素c荧光标记物存在于青年组大鼠耳蜗基膜毛细胞质（细胞膜的内侧，线粒体存在部位），增强的细胞色素C免疫反应物存在于老年组大鼠耳蜗基膜细胞。细胞色素c阳性反应见于：①细胞核型不变；②碘化丙锭染色略有加深，细胞核型不变；③细胞核轻微核固缩；④核肿胀的毛细胞。相反，部分核固缩≤3／4核的毛细胞细胞色素C阳性反应物减少或消失，核缺失区域的毛细胞未见细胞色素c阳性反应物。老年大鼠耳蜗基膜凋亡毛细胞线粒体释放的细胞色素c存在核内转移现象。结论：细胞色素c免疫荧光染色为老年耳蜗基膜毛细胞退行性变早期的细胞生物学标记之一。细胞色素C向细胞核内的转移，可能与老年大鼠耳蜗凋亡毛细胞染色质凝聚相关。

Changes of cytochrome C expression at different stages of apoptotic and necrotic hair cells in the cochleae of aging Sprague-Dawley rat

Objective：To investigate the mechanism of hair cells death in the aging rat cochleae, we observed changes of cytochrome C expression at different stages of apoptotic and necrotic hair cells. Method： A total of 16 Sprague-Dawley（SD） rats were used in present study. The animals were assigned into two groups： the young rat group, ages 2--3 months（n=8） and the aging rat group, ages 24--26 months（n=8）. The auditory brainstem re- sponse（ABR） thresholds of both ears elicited by tone bursts at 4, 8, 16, 32 and 40 kHz were measured. When finished the auditory test, cochleae were quickly removed from the skull. Whole specimens comprising the basilar membranes with Corti＇s organs were separated from the modiolus. The organs of Corti were double stained with propidium iodide（PI）, a DNA intercalating fluorescent probe used to visualize the morphologic viability of hair cell nuclei, and cytochrome C was performed to visualize the release of cytochrome C from the mitochondria in hair cells, every organ of Corti was thoroughly examined using fluorescence microscopy. Result：Aging rats exhibited a significant elevation of ABR thresholds at all tested frequencies（P〈0. 01）. Cochleae from young showed slight cy- tochrome C fluorescent in the cytoplasm along the inner surface of the plasma membrane where mitocbondria nor- mally reside in hair cells. Strong cytochrome C fluorescent labeling was seen in some hair cells in the aging cochle- ae. positive reactions of cytochrome C fluorescence were present in hair cells with normal nuclei, the nuclei with an increase PI fluorescence but without marked shrinkage, mild nuclear condensation, nuclear swelling. In contrast, the positive reactions of cytochrome C staining was markedly reduced or absent in some hair cells with slight nucle-ar condensation （about≤3/4 of the normal size）. Finally, in the areas where no detectable nuclear staining was present, cytochrome C fluorescent was absent. Some apoptotic hair cells showed nuclear cytochrome C aggrega- tion, which may be a phenomenon for nuclear redistribution of cytochtome C. Conelusion： These results suggest that release of cytochrome C from the mitochondria is one of early biological marks for hair cells degeneration in aging cochlea. Nuclear translocation of cytochrome C during apoptosis may be involved in chromatin condensation in age-related hair cells death.