顺铂对离体培养小鼠耳蜗毛细胞的损伤及凋亡作用

Effect of Cisplatin on Impairment and Apoptosis of Mouse Cochlear Hair Cells in Vitro

目的建立新生昆明小鼠离体顺铂耳毒性模型,研究顺铂对离体培养小鼠耳蜗毛细胞的损伤及凋亡作用。方法分离生后3d的昆明小鼠耳蜗基底膜,在含有1%牛血清白蛋白的DMEM/F12培养基中进行培养。采用异硫氰酸四甲基罗丹明标记的鬼笔环肽荧光染色方法观察不同浓度顺铂对小鼠耳蜗毛细胞的损伤,同时应用Hoechst 33258染色技术检测耳蜗毛细胞的凋亡。结果不同浓度顺铂均可使小鼠耳蜗毛细胞发生凋亡的形态学变化。顺铂浓度为4mg·L-1时,耳蜗毛细胞缺失率和凋亡率明显增大,与对照组比较差异有统计学意义(P<0.01)。随着顺铂浓度的增高(8～64mg·L-1),耳蜗毛细胞缺失率和凋亡率亦显著增大,呈现明显的量效关系(P<0.01)。结论应用新生昆明小鼠能建立可靠的离体顺铂耳毒性模型;顺铂可导致耳蜗毛细胞凋亡,提示凋亡可能是顺铂耳毒性机制之一。

Objective To establish a newborn Kunming mouse model of cisplatin-induced ototoxicity in vitro,and to investigate the effect of cisplatin on impairment and apoptosis of cochlear hair cells.Methods The cochlear basilar membrane was isolated from mice at postnatal day 3 and cultured in DMEM/F12 medium containing 1% bovine serum albumin.Tetramethylrhodamine isothiocyanate-labeled phalloidine staining was used to observe the impairment effects of different concentrations of cisplatin on mouse cochlear hair cells,and Hoechst 33258staining was used to detect cell apoptosis.Results Apoptotic changes in cochlear hair cells were induced by different concentrations of cisplatin.Compared with the control,the percentage of missing and apoptotic hair cells was significantly increased by cisplatin at a concentration of 4mg·L-1（P〈0.01）.With the increase of cisplatin concentration（8～64mg·L-1）,the percentage of missing and apoptotic hair cells was significantly increased,showing a clear dose-response relationship（P〈0.01）.Conclusion A Kunming mouse model of cisplatin-induced ototoxicity in vitro can be established.Cisplatin can lead to apoptosis of cochlear hair cells,suggesting that apoptosis may be one of the mechanisms of cisplatin ototoxicity.