摘要
目的通过检测3种半胱氨酸蛋白酶(caspase-3、caspase-8和caspase-9)在耳蜗基底膜的活性变化,揭示强脉冲噪声暴露后耳蜗毛细胞死亡的信号通路。方法成年灰鼠27只,随机分为实验组和对照组。接受噪声暴露的实验组动物共15只,3种半胱氨酸蛋白酶荧光标记动物各5只。未经噪声暴露的对照组动物共12只,3种半胱氨酸蛋白酶荧光标记动物各4只。将动物暴露于平均压力峰值级为155dB SPL的脉冲噪声75次,噪声暴露后2h,用微量注射器分别将新鲜配制的3种半胱氨酸蛋白酶荧光染色液30μl缓慢注入动物双侧内耳。耳蜗内灌注后1h,动物断头,取出双耳听泡,分离耳蜗基底膜。采用细胞核DNA荧光染料碘化丙锭(PI)对耳蜗基底膜毛细胞核进行染色,荧光显微镜下观察强脉冲噪声暴露后的耳蜗基底膜凋亡、坏死毛细胞3种半胱氨酸蛋白酶荧光染色的变化。结果对照组灰鼠的耳蜗毛细胞核周围未见3种半胱氨酸蛋白酶阳性荧光标记物。实验组噪声暴露后耳蜗基底膜以核固缩、核碎裂为特征的凋亡外毛细胞核周围存在caspases-3、caspases-8和caspases-9绿色荧光标记物,而以核肿胀为特征的坏死外毛细胞核周围未见3种半胱氨酸蛋白酶阳性荧光标记物。结论强脉冲噪声暴露后耳蜗毛细胞凋亡同时通过caspases-9相关的内源性和caspases-8相关的外源性两条信号通路启动完成,而毛细胞坏死则是通过非caspases途径激活的。
Objective Apoptosis and necrosis are two forms of hair cell death in cochlea following exposure to intense impulse noise. The present study was designed to examine the activity of caspase-3, caspase-8 and caspase-9 in the basilar membrane of cochlea, and investigate the cellular signal pathway associated with noise-induced hair cell death in cochlea. Methods Twenty-seven adult chinchillas were used in present study. The animals were randomly assigned into test group （no. 15） and control group （no. 12）. The animals in test group were exposed to noise for the detection of the activity of easpase-3, caspase-8 and easpase-9, respectively, each with 5 animals. Animals in control group were not exposed to noise. Chinchillas were exposed to impulse noise at 155 dB peak sound pressure level （pSPL） for 75 times. Two hours after noise exposure, both cochleae were perfused with approximately 30μl of freshly prepared solution containing one of the three caspases （easpase-3, 8 and -9） respectively using a micro-syringe. One hour after perfusion, the animals were sacrificed and the cochleae were harvested. The Corti organs were double stained with propidium iodide （PI）, and a DNA intercalating fluorescent probe was used to visualize the morphologic viability of hair cell nuclei. All the specimens were observed with a fluorescence microscope. Results The normal cochlea did not exhibit green fluorescence for any of the three caspases in Corti organ. Strong easpase-3, -8 and -9 green fluorescence appeared in the outer hair cells with condensed or fragmented nuclei, a sign of apoptosis, whereas no fluorescence was observed for any of the three easpases in the swollen outer hair cell nuclei, a sign of necrosis, in the cochleae exposed to intense impulse noise. Conclusion It is indicated that caspase-9 is involved in the intrinsic and easpase-8 involved in the extrinsic apoptotic pathway which occur sim uhaneously in apoptotie outer hair cells. No caspase activation occurs in necrotic outer hair cell in the cochlea following exposure to intense impulse noise.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第1期95-97,共3页
Medical Journal of Chinese People's Liberation Army
基金
美国NIH资助项目(NIDCD1R03DC006181-01A1)
