内耳感觉细胞自噬与内质网应激在顺铂耳中毒损伤中的作用及机制研究

Cisplatin is one of the most widely used chemotherapeutic agent. However, side effects like nephrotoxicity, ototoxicity and bone marrow toxicity have restricted its clinical applications. However ,there is no effective treatment for the disease. Autophagy is a conserved widespread bulk intracellular degradation system that eliminating damaged long-lived proteins and organelles in eukaryocytes, which protect the cells from disadvantaged circumstances. A large number of studies have suggested that inducing autopahgy properly and modulating of endoplasmic reticulum stress could protect organs from pathologic factors,and maintain normal functions. Our previous experimental study have demonstrated that autophagy induced by rapamycin may attenuate oxidative damage of the inner ear caused by cisplatin in rats, which may guards against cisplatin ototoxicity. However, the mechanism is still not clear. Based on our previous study ,we will apply molecular biological methods like RT-PCR, Western blot. As well as electrophysiological , morphological study to determine: 1. the protective effects of autophagy guards against the cisplatin induced oxidative stress in the spiral ganglion cells,the hair cells and the marginal cells; 2.To investigate the dose-effcet relationship of the rapamycin induced autophagy and protective response in the GFP-LC3 transgenic mice,the HEI-OC1 hair cell line insulted with cisplatin; 3. In vitro and in vivo studies will explore the role of endoplasmic reticulum played in cisplatin-induced ototoxicity models; 4. To explore the mechanisms of calcium ion channels of the membrane modulating the process of mitophagy in the spiral ganglion cells and the hail cell lines. The findings of this work would be the basis for investigating role of autophagy and endoplasmic reticulum stress in cisplatin ototoxicity models, which may provide a novel therapeutic option to minimize cisplatin-induced ototoxicity.

顺铂是临床常用化疗药物，它造成的耳毒性、肾毒性及骨髓毒性限制了其临床应用。目前仍缺乏有效的治疗手段。自噬是真核细胞广泛存在的不利环境下清除胞内蛋白质和受损细胞器的自身保护机制。大量的研究证实，适度诱导自噬及调控内质应激可有效对抗各种病理损伤，维护脏器功能。我们前期的实验研究表明雷帕霉素诱导自噬可以显著减少顺铂造成的内耳氧化损伤，拮抗顺铂耳毒性，但机制不明确。本课题拟在此基础上利用RT-PCR，蛋白印迹等分子生物学技术及形态学、细胞电生理等方法研究：1.探讨诱导自噬对毛细胞、螺旋神经节细胞及边缘细胞的保护作用；2.探明不同浓度雷帕霉素诱导自噬与降低氧化损伤的量-效关系；3.探明内质网应激在顺铂耳中毒损伤中调控作用；4.探讨毛细胞、螺旋神经节细胞钙离子及钾离子通道对自噬的调节机制。本课题研究结果将阐明内耳感觉细胞自噬与内质网应激在顺铂耳中毒损伤的作用和机制，为顺铂耳毒性的防治提供新的策略。

顺铂耳中毒性聋是目前影响接受顺铂化疗患者生存质量的重要原因之一，其发生机制至今尚未被完全阐明，临床上亦缺少可靠的防治手段。通过本项目的研究，我们发现：①内质网应激及其相关凋亡可能是顺铂对内耳感觉细胞毒性损伤发生的重要分子机制之一。通过应用内质网蛋白质稳态保护剂牛磺酸熊去氧胆酸（TUDCA）可以有效拮抗顺铂耳中毒性聋的发生。②靶向细胞自噬的上游信号GSK3β和sirt3，可实现对内耳感觉细胞中细胞自噬的有效诱导，并最终实现对顺铂致内耳感觉细胞毒性损伤的拮抗。本项目的上述发现进一步阐明了顺铂致内耳感觉细胞毒性损伤的分子机制，为临床上顺铂耳中毒性聋防治新策略的制定提供了实验基础。