ROS-Caspase-3-Dicer通路在钴诱导耳蜗细胞毒性损伤中的作用及分子机制

医源性钴暴露和环境钴暴露可引起耳神经毒性损伤，但具体机制尚不明确。我们前期研究中，成功构建钴中毒的动物体内、体外模型，并发现钴离子能诱导内耳毛细胞和螺旋神经节细胞中的有害氧自由基累积和Caspase-3表达增加，并造成细胞凋亡。已有研究表明，用氯化钴诱导细胞缺氧后，活化的Caspase-3能降解miRNA加工酶Dicer，诱导细胞死亡。据此，结合我们已有的研究，本项目提出以下科学假设：钴的内耳神经损伤作用可能与活性氧激活Caspase-3通路、降解Dicer导致细胞凋亡有关。本项目拟用荧光免疫组化、高通量测序、蛋白芯片等技术研究钴处理前后内耳毛细胞和神经纤维凋亡等情况，从基因、蛋白水平检测钴对听觉及前庭神经系统损害过程中信号通路发生的系列变化；同时应用siRNA技术干扰Caspase-3-Dicer通路的关键分子表达，探讨该通路对钴耳毒性作用的影响，并为干预措施提供潜在的调控新靶

Studies have shown that endogenous exposure of the implants made of cobalt-titanium alloy and environmental cobalt exposure can lead to symptoms of ototoxicity. Cobalt poisoning may result in the occurrence of hearing loss, though the mechanism has yet to be explained. Our previous studies had shown that Cobalt ions was corresponding to the accumulation of oxygen free radicals and the expression of Caspase-3 in the cochlear hair cells and ganglion cells. It has been reported that activated Caspase-3 can reduce the level of Dicer and induce cell death after simulating cell hypoxia with CoCl2. Based on this, we propose a scientific hypothesis: the damage effect of cobalt on the inner ear nerve may be related to the activation of Caspase-3 signaling pathway by reactive oxygen species and the reduction of Dicer level leading to cell apoptosis. Based on the previous work, we intend to use the successfully built model of cobalt-induced hearing loss in rat to study the autophagy phenomenon and changes of gene and protein expression of the cochlear hair cells, nerve fibers and spiral ganglion cells before and after cobalt intervention. At the same time, siRNA will be used to interfere with the expression of key molecular in the pathway of Caspase-3-Dicer, in order to detect cobalt induced damages in molecular level.