庆大霉素对小鼠耳蜗螺旋神经节细胞损伤机制初步研究

目的探讨庆大霉素对小鼠耳蜗螺旋神经节细胞（spiral ganglion cell,SGC）的损伤机制。方法取出生后2～6天昆明种乳小鼠耳蜗,利用酶消化和机械分离相结合的方法分离出SGC,并进行体外培养。将培养4天的SGC用4%多聚甲醛室温固定30min,以小鼠源神经纤维细丝蛋白（Neurofilament-68/200kDa,NF-L＋H）单克隆抗体作为一抗,按照SP法（streptavidin-perosidase法,链霉菌抗生物素蛋白-过氧化物酶连结法）对所培养细胞进行免疫细胞化学染色,对SGC作出鉴定。将培养第4天的细胞分为4组：空白对照组及3种浓度的庆大霉素干预组（庆大霉素终浓度分别为50mg/L、100mg/L、150mg/L）,作用48h后收集各组细胞进行透射电镜分析。结果SGC原代培养获得成功,经NF-L＋H抗体免疫细胞化学染色,其胞质及突起均呈阳性反应,染成棕黄色,一般有相对生长的两个突起,为典型的双极神经元。在透射电镜下观察SGC超微结构,3种浓度的庆大霉素组SGC与空白对照组相比,均出现明显的形态学改变,这种改变与凋亡相关。结论庆大霉素对小鼠耳蜗SGC有直接毒性作用,可导致细胞凋亡,线粒体损伤在这一过程中可能具有重要意义。

Injury mechanisms of cultured mouse spiral ganglion cells by gentamicin

Objective To study in vitro injury mechanisms in spiral ganglion cells (SGC) of postnatal mouse cochlear caused by gentamicin. Methods SGCs were isolated using a combinatorial approach of enzymatic digestion and mechanical separation from cochleae of P2-6 Kunming mouse. After 4 days, cultured SGCs were fixed with 4 % paraformaldehyde in room temperature and then labeled with the monoclonal antibody to mouse neurofilament protein (Neurofilament-68/200 Kda, NF-L+H) using S-P method for immunocytochemical identification. SGCs were then ran-domly divided into a control group and three test groups that were treated with gentamicin at 50 mg/L, 100 mg/L and 150 mg/L. After 48 hours of incubation, SGCs were examined under a transmission electron microscope. Results The SGCs were successfully cultured and their cytoplasm and neurites were labeled brownish yellow with the monoclonal antibody to mouse neurofilament protein, demonstrating classical bipolar growth patterns with two opposing neurites. Compared to the control group, gentamicin-treated SGCs showed morphologic changes indicating apoptosis. Conclusion Gentamicin has direct toxic effect on SGCs from mouse cochlear and the injury mechanism is closely concerned with apoptosis. The dam-age of mitochondria may play an important role in the process.