耳蜗细胞死亡方式的鉴别  

Identification of cochlear cell death

作　　者：丁大连[1,2,3] 李鹏[2,3] 高可雷 蒋海燕 孙虹[1,3] 陈成芳[4] Richard Salvi 

机构地区：[1]中南大学湘雅医院耳鼻咽喉头颈外科,湖南长沙410008 [2]中山大学附属第三医院耳鼻咽喉头颈外科,广东广州510630 [3]Center for Hearing and Deafness University at Buffalo [4]山东省立医院耳鼻咽喉科,山东济南250014

出　　处：《中国耳鼻咽喉颅底外科杂志》2015年第3期178-183,共6页Chinese Journal of Otorhinolaryngology-skull Base Surgery

摘　　要：目的通过几种不同耳毒性药物离体培养实验和活体动物实验,以观察耳蜗细胞死亡的不同方式。方法应用Apoptosis-Necrosis Quantification Kit对正常或耳毒性药物处理过的离体培养耳蜗组织和活体耳蜗组织进行了染色。试剂盒中含有3种不同荧光标记的探针,其中绿色荧光标记的annexin V可鉴别活细胞内出现的凋亡改变,红色荧光标记的Ethidium homodimer III可鉴别活细胞内出现的坏死改变,蓝色荧光标记的Hoechst 33342则可鉴别出健康细胞。结果在耳蜗离体培养实验中,细胞凋亡信号和坏死信号几乎同时出现在顺铂或庆大霉素处理的耳蜗外植体;在活体动物实验中,细胞凋亡信号和坏死信号也同时出现在急性卡那霉素损害模型和慢性卡那霉素损害模型。结论在细胞受损后的变性过程中,凋亡因子和坏死因子可同时出现而导致细胞的死亡。细胞凋亡的过程一般比较迅速而常常发生在急性损害阶段,而细胞坏死的过程往往比较缓慢而多见于细胞的慢性损害过程。在细胞死亡的最后阶段,细胞的最终破坏一般取决于哪种死亡方式抢先完成,但无论是哪种死亡方式占据了主导地位,都不能排除另外一种细胞死亡方式对细胞降解过程的协同作用。Objective We identified cochlear cells undergoing apoptosis and necrosis in vitro and in vivo after treatment with several ototoxic drugs. Cochlear explants were treated with gentamicin（ 0. 5 m M） for 24 h or cisplatin（ 50 μM） for 48 h. In adult rats,acute cell death was investigated after co-administration of kanamycin（ KM,800 mg/kg,I. M.） plus ethacrynic acid（ EA,50 mg / kg,I. V.）; chronic cell death in adults rats was investigated by treating adult rats for 8 d with daily injection of KM（ 800 mg / kg,I. M.）. Methods Cochlea hair cell death was identified using an Apoptosis-Necrosis Quantification Kit. Apoptotic cells were identified by annexin V conjugated with FITC（ green fluorescence） which specifically binds to the phosphatidylserine exposed on the outer membrane leaflet. Necrotic cells were identified by labeling with ethidium homodimer III（ Eth D-III,red fluorescence）,a highly charged nucleic acid probe.Healthy cells were labeled with the membrane-permeant blue fluorescent DNA dye,Hoechst 33342. Results In vitro studies,both apoptotic and necrotic signals were detected in cisplatin treated cochlear cultures and also in gentamicin treated cochlear explants. In vivo studies,apoptotic and necrotic signals were also found in degenerating hair cells in both acute damage model and chronic damage model. Conclusions In the process of cell degeneration,both apoptotic factors and necrotic factors can be activated leading to the cell death. Apoptotic process is relatively fast which often occurs in acute damage. In contrast,necrotic process is relatively slow which usually involves chronic damage. In the final moment of cell death,cell destructions may depend on which death way is preemptive fatal. However,no matter which death way dominates,we cannot rule out the synergistic effects on cell degradation by other death pathway.

关 键 词：耳蜗 细胞凋亡 细胞坏死 细胞死亡 耳毒性药物 

分 类 号：R764[医药卫生—耳鼻咽喉科]