硫酸链霉素引起的大鼠体外培养耳蜗毛细胞凋亡

目的 探讨在离体培养条件下硫酸链霉素是否通过半胱氨酸天冬氨酸蛋白酶(caspase)的激活而导致大鼠耳蜗毛细胞凋亡.方法 将出生后3～4 d的大鼠耳蜗基底膜取出进行离体培养,培养过夜后用0.1 mmol/L硫酸链霉素培养液继续培养24 h.终止培养前1 h应用碳氧荧光素标记的肽荧光甲基酮(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)标记的caspase-6,caspase-8,或caspase-9指示剂作用1 h,然后应用异硫氰酸四甲基罗丹明(tetramethyl rhodamine iso-thiocyanate,TRITC)标记的鬼笔环肽(phalloidin)标记毛细胞的纤毛和表皮板,再应用Topro-3 DNA探针对细胞核进行染色,最后在共聚焦显微镜下观察.结果 1 mmol/L硫酸链霉素引起的耳蜗毛细胞缺失在底回约为80%,在顶回约为20%.经链霉素作用后,耳蜗毛细胞的细胞核发生浓缩和碎裂,同时可见cmpase-6,caspase-8和caspase-9阳性表达.结论 硫酸链霉素可以引起离体培养的大鼠耳蜗毛细胞凋亡,上游和下游的caspase激活可能是其主要途径.

Streptomycin-induced apoptosis of rat cochlear hair cell cultured in vitro

Objective To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells. Methods F344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope. Results Streptomycin with 1 nunol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated. Conclusions Apoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.