顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶表达的影响

目的：观察顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶（calpain ）表达的影响，探讨顺铂致耳蜗毛细胞凋亡的机制。方法取出生后3 d的昆明小鼠300只（600耳），分离出耳蜗基底膜600条，体外培养24 h后，随机分为对照组和4、8、16μg／ml顺铂组，每组150条；对照组加入2ml新鲜培养基，顺铂组分别加入2 ml含不同浓度顺铂（4、8、16μg／ml）的新鲜培养基，再继续培养24 h后，应用Hoechst 33258荧光染色观察耳蜗毛细胞凋亡情况，并应用免疫荧光染色和免疫印迹（Western blot）技术检测calpain 1（μ－calpain）和calpain 2（m －calpain）在耳蜗毛细胞的表达。结果4、8、16μg／m l顺铂组耳蜗毛细胞凋亡率分别为15．63％±0．20％、38．40％±2．64％和64．24％±0．05％，均高于对照组（5．55％±0．12％），呈现明显的量效关系；不同浓度顺铂组μ－calpain和m －cal‐pain的表达均较对照组明显增强（ P＜0．01），且m －calpain的表达随顺铂浓度的增高而明显增强（ P＜0．01）。结论顺铂可通过calpain通路诱导小鼠耳蜗毛细胞凋亡，从而发挥毒性效应。

The Effects of Cisplatin on the Expression of Calpain in Mouse Cochlear Hair Cell in Vitro

Objective To investigate the effects of cisplatin on the expression of calpain in mouse cochlear hair cells in vitro ,and to explore the mechanism of cisplatin-induced apoptosis in mouse cochlear hair cells .Meth‐ods A total of 600 cochlear basilar membranes isolated from Kunming mice at postnatal day 3 were cultured for 24 hours ,then randomly divided into control group and 3 cisplatin groups (4 μg/ml ,8 μg/ml and 16 μg/ml) .Each group contained 150 basilar membranes .Four groups were continually cultured for another 24 hours .Hoechst 33258 staining was used to detect the apoptosis of cochlear hair cell .Immunofluorescent staining and Western blot were carried out for detecting the expressions of calpain 1 (μ-calpain) and calpain 2 (m-calpain) in mouse cochlear hair cells .Results The percent of apoptotic hair cells in the three cisplatin groups (15 .63% ± 0 .20% ,38 .40% ± 2 .64% and 64 .24% ± 0 .05% ,respectively) was greater than that of in the control group (5 .55% ± 0 .12% ) , showing a clear dose-response relationship (P<0 .01) .Furthermore ,the expressions of μ -calpain and m -cal‐pain in different cisplatin groups were increased ,and m -calpain expression was great remarkably with increased concentration of cisplatin (P<0 .01) .Conclusion Our results suggest that cisplatin can induce the apoptosis in mouse cochlear hair cells by regulating calpain pathway .This may play a role in the cisplatin-induced ototoxicity .