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目的:探讨强噪声对豚鼠耳蜗细胞死亡的机制及磷酸化c-Jun氨基末端激酶(JNK)信号通道在强噪声诱导耳蜗细胞凋亡的作用。方法:将实验组豚鼠暴露在4kHz窄带噪声120dBSPL噪声环境中4h,噪声刺激停止后1d、4d、14d组及对照组(每组各8只)在处死前测ABR。取每组4只豚鼠耳蜗作石蜡切片,另外4只豚鼠提取耳蜗总蛋白。脱氧核糖核甘酸末端转移酶介导的缺口末端标记技术(TUNEL)检测耳蜗凋亡细胞,免疫组织化学及WesternBlot方法检测JNK信号途径蛋白质P-JNK、P-c-Jun的表达。结果:实验组耳蜗Corti器毛细胞、血管纹和螺旋神经节细胞存在TUNEL阳性细胞,以1d组最多,逐渐减少,14d组最少,而对照组未见阳性细胞。免疫组织化学观察到实验组P-JNK、P-c-Jun有免疫反应阳性,定位于细胞核,对照组未见阳性细胞。Western Blot检测P-JNK、P-c-Jun含量在噪声刺激后迅速增高并快速活化,1d、4d达到高峰,随后逐渐下降,但在14d仍然维持较高水平。结论:强噪声可以通过诱导凋亡造成耳蜗细胞损伤,同时P-JNK标志着JNK信号途径的激活,提示JNK信号通道可能也是介导强噪声诱导豚鼠耳蜗细胞凋亡信号通道之一。
Objective: To investigate the mechanism of strong noise on cochlear cell death in guinea pigs and the effect of phosphorylated c-Jun N-terminal kinase (JNK) signaling pathway on cochlear apoptosis induced by strong noise. Methods: The guinea pigs in experimental group were exposed to noise of 120dBSPL at 4kHz for 4h, and ABR was measured at 1d, 4d, 14d and 8th after the cessation of noise stimulation. Four cochlear guinea pigs in each group were paraffin-embedded and four cochlear guinea pigs were used for total cochlear protein extraction. Cortical apoptotic cells were detected by TUNEL. The expressions of P-JNK and P-c-Jun proteins were detected by immunohistochemistry and Western Blot. Results: There were TUNEL positive cells in the hair cells, stria vascularis and spiral ganglion cells in the Corti hair cells of experimental group. The number of TUNEL positive cells was the highest in group 1d and the lowest in group 14d, but not in control group. Immunohistochemistry showed that the experimental group P-JNK, P-c-Jun immunoreaction positive, located in the nucleus, the control group no positive cells. The content of P-JNK in P-JNK was detected by Western Blot. The content of P-c-Jun increased rapidly and rapidly after noise stimulation, peaked at 1d, 4d, then decreased gradually, but remained high at 14d. CONCLUSION: Strong noise can induce cochlear cell injury by inducing apoptosis, and P-JNK marks the activation of JNK signaling pathway, suggesting that JNK signaling pathway may also be one of the signal transduction pathways that induce strong noise-induced cochlear cell apoptosis in guinea pigs.