【作者】 原红艳；

【导师】 李树华；

【作者基本信息】 大连医科大学 ， 耳鼻咽喉科学， 2005， 硕士

【摘要】 过量的活性氧（reactive oxygen species, ROS 或称氧自由基;oxygen-free radical,OFR）如O² -、H₂O₂、OH-、脂质过氧化物等可引起不同程度的细胞毒性,或导致瞬时的或不可逆的损伤。细胞凋亡是多种生理病理因子参与的由凋亡相关基因启动的细胞凋亡过程,许多炎症介质和细胞因子均影响细胞凋亡过程。其中,氧化应激造成的大量ROS 的产生及其迟发性在细胞凋亡过程中起重要作用。大量研究表明,药物性聋、噪声性聋、缺血再灌注性聋及老年性聋与ROS 增多密切相关,且在这些内耳疾病中ROS 均参与了耳蜗毛细胞凋亡、死亡的过程。因此在听力损伤机制中,ROS 中毒导致毛细胞凋亡的机制具有重要意义。以往有学者通过在体的噪声性聋、顺铂及氨基糖苷类抗生素等药物性聋等实验模型间接推测ROS 可诱导毛细胞凋亡,但利用ROS 的直接损伤模型来观察细胞凋亡尚鲜有报道。目前ROS 通过何种途径引起耳蜗毛细胞缺失尚未完全阐明,因此研究ROS 诱导的耳蜗毛细胞凋亡及其机制,寻求高效低毒的天然抗氧化剂及设法调动或激活机体中的内源性抗氧化剂,将为感音神经性耳聋的药物干预措施提供依据。本研究则利用耳蜗Corti 器体外培养技术,建立一种离体耳蜗的ROS 损伤模型;检测ROS 引起的耳蜗毛细胞凋亡; N-乙酰半胱氨酸（n-acetylcysteine, NAC）是一种含巯基化合物,它作为体内活性氧系列物质（如H2O2和O2-）的清除剂和还原性谷胱甘肽GSH 的供给体,在机体抗氧化反应中起到重要作用。NAC 还可对抗多种损伤导致的细胞凋亡,因此我们观察了NAC 对ROS 诱导的耳蜗毛细胞凋亡的抑制更多还原

【Abstract】 Excessive oxygen free radical(sOFR)may produce severe cell toxicity, they also can cause the instant or irreversible damage. The cell apoptosis is a process that involves many kinds of physiological and pathological factors, and many inflammation media and cell factors all affect the process. It is very important that the production of massive OFR and the late response induced by oxidative stress for the process of cell apoptosis. Many studies have indicated that the deafness resulting from drug toxicity、noise-induced hearing loss 、ischemical reperfusion deafness and presbycusis all have relationship with OFR. In these deseases ,ROS is all been invovled and it may cause apoptosis or necrosis of cochlea hair cell. So the mechanism of OFR-induced apoptosis on hair cell has very important significance. It has been suspected that OFR can induce apoptosis in many experiments in vivo, but the apoptosis model directly induced by OFR in vitro hasn’t been seen. It is still not clear that the way caused the cochlea hair cell depletion. To seek for effective natural antioxidants and try to activate endogenous antioxidants will provide evidence for the treatmen of sensorineural hearing loss. In this experiment ,we set up a damage model of OFR by tissue culture methods of organ of Corti in neonatal S.D. rat. The apoptotic hair cells of cochlea were detected. N-acetylcysteine (NAC) works in the extracellular environment, and is a free radical scavenger and a precursor of intracellular cysteine and glutathione (GSH). NAC prevent apoptosis caused by many kinds of injury. In this study, the inhibit effects of NAC on OFR-induced apoptosis were studied in vitro. The study is divided into the following three parts. Part one: To set up tissue culture methods of organ of Corti in neonatal S.D. rat and investigate the application of AO/PI staining on the detection of cochlea hair cell. Twenty-four hours after the tissue cultivation, new epithelia cells and fibroblast were found around the tissue. Inner hair cells(IHCs) 、outer hair cell(OHCs) and supporting cell grew well. The tissue was stained by Acridine orange(AO) and Prodium iodine(PI). It becomes green,which means the tissue has good activity. The culture of organ of corti can be an ideal method for otology research. Part two: To observe the apoptosis on organ of Corti induced by OFR in vitro. Forty-eight hours after the tissues cultivation, the tissues was stained by AO and PI. We observed the ratio of apoptotic cells in difffent groups. There was a higher ratio of apoptotic cells on basal outer hair cells compared with apical hair cells and middle hair cells. In every group, OHCs had a higher ratio of apoptotic cells than IHCs. Our results suggest OFR can cause apoptosis of hair cells, and the way it induced cochlea damage is dose-dependent furthermore. The basal hair cells may be more vunerable to OFR damage than apical hair cells. OHC are more susceptible to OFR than IHC. Supporting cells have considerably more survival capacity than sensory cells. It is possiblely because of the Ca2+ overload and the decrease of anti-oxidized ability that H2O2 induced the apoptosis on hair cell. Part three: To investigate the inhibit effect of NAC on OFR-induced apoptosis of cochlea hair cells. We observed the ratio of apoptotic cells in difffent groups. The ratio of apoptotic cells was significantly decreased by addition of the radical scavenger NAC. The protection by antioxidants implies that the apoptosis of hair cells is duo to OFR damage.In conclusion, the culture of organ of corti can be an ideal method for otology research. OFR can induce apoptosis of hair cells. The morphological change is consistent with free-radical mechanisms involved in the ototoxic side effects of aminoglycosides and noise-induced hearing loss. The effectiveness of antioxidants in improving the viability of hair cells indeed supports free-radical mechanisms as the ditermining cause of cell apoptosis. The biochemical basis of this phenomenon, susceptibility to free radicals, may offer further insight into ot更多还原