中文摘要：

目的通过观察耳蜗基底膜毛细胞凋亡相关因子caspase-3、caspase-8和caspase-9表达的变化，揭示老年大鼠耳蜗毛细胞凋亡的信号通路。方法人选Fischer344大鼠27只．青年组12只，3-4月龄；老年组15只，20～27月龄。3种半胱氨酸天冬氨酸蛋白酶荧光标记青年大鼠耳蜗各8个，老年大鼠耳蜗各10个。应用电位反应测听仪检测不同频率短纯音（5、10、20和d0kHz）诱发的青年和老年大鼠双侧听性脑干诱发电位反应阈值（ABR）。听觉功能测试后，用微量注射器分别将新鲜配制的3种半胱氨酸天冬氨酸蛋白酶荧光染色液缓慢注入动物内耳，耳蜗内灌注后1h，动物断头处死。采用细胞核DNA荧光染料碘化丙锭（PI）染色耳蜗基底膜细胞，荧光显微镜下观察不同月龄大鼠耳蜗基底膜毛细胞3种半胱氨酸天冬氨酸蛋白酶荧光染色的变化。结果老年大鼠的不同频率听性脑干反应阈值均高于青年对照组（P〈0．05）。青年大鼠耳蜗毛细胞核周围未见3种半胱氨酸天冬氨酸蛋白酶阳性荧光标记物，老年大鼠耳蜗基底膜以核固缩为特征的凋亡外毛细胞核周围存在caspases-3和caspases-9绿色荧光标记物，未见caspases-8标记物。以核肿胀为特征的坏死外毛细胞核周围未见3种半胱氨酸天冬氨酸蛋白酶阳性荧光标记物。结论老年大鼠耳蜗毛细胞凋亡可能通过caspases-9相关的线粒体信号转导通路而不是easpases～8相关的死亡受体通路启动完成。

英文摘要：

Objective To examine the expression of apoptosis-related factors caspases-3, caspases-8 and caspases-9 in the basilar membrane of the cochlea and investigate the cellular signal pathways associated with age-related hair cell death in the cochlea. Methods Twenty-seven Fischer 344 rats were used in this study, including 12 young （3-4 months old） and 15 aging （20-27 months old） animals. Auditory brainstem response （ABR） thresholds were determined with tone bursts at 5, 10, 20 and 40 kHz. Upon completion of the auditory test, cochleae were perfused with approximately 30 p.1 of a freshly prepared solution containing caspase-3, -8 or -9 using a micro-syringe （8 cochleae each in young animals and 10 cochleae each in aging animals）. One hour after perfusion, animals were sacrificed and cochleae harvested. The organs of Corti were double stained with propidium iodide （PI）, a DNA intercalating fluorescent probe used to visualize the morphologic viability of hair call nuclei. Specimens were examined under a fluorescence microscope for activities of caspase-3, -8 and -9. Results Aging rats exhibited a significant elevation of ABR thresholds at all tested frequencies （P 〈 0.05）. Cochleae from young rats showed no green fluorescence for any of the three easpases in the organs of Corti. Strong caspase-3 and -9 fluorescence was seen in outer hair cells with condensed or fragmented nuclei, a assign of apoptosis, without signs of caspase-8 fluorescence, whereas no fluorescence was seen for any of the three caspases in swollen outer hair cell nuclei, a sign of necrosis. Conclusion The results of the study indicate that caspase-9 is involved in the intrinsic apoptotic pathway in outer hair cells in the aging cochlea. In contrast, caspase-8 activity was not significantly affected by age.