锰诱导体外培养耳蜗毛细胞的损伤与凋亡研究

Manganese-induced Damage and Apoptosis of Cochlear organotypic cultures

目的探讨锰诱导体外培养耳蜗毛细胞损伤与凋亡的关系。方法将出生3天的新生Sprague Dawley大鼠的耳蜗器官体外培养,将贮存浓度为100m M氯化锰用SFM稀释到终浓度为1m M,3m M,5m M后进行体外染毒处理耳蜗基底膜。应用鬼笔环肽染色特异性显示耳蜗毛细胞的静纤毛,同时用β-Tubulin对神经纤维进行染色,用TUNEL试剂盒;Cleaved caspase-3抗体对耳蜗基底膜进行染色,在激光共聚焦显微镜下分别观察耳蜗基底膜的毛细胞,神经纤维。结果耳蜗基底膜培养24小时,1m M氯化锰对耳蜗毛细胞及神经纤维损伤甚微,差异无统计学意义(P>0.05),内,外毛细胞排列整齐规律,听神经纤维分布均匀,成束排列。最高浓度5m M氯化锰处理24小时后对神经纤维造成明显的损害,与对照组相比,差异有统计学意义(P<0.05)。当浓度逐渐增加,损伤愈加显著并呈浓度依赖性。2m M氯化锰处理,对耳蜗基底膜底转损伤较中转、顶转明显。不同浓度氯化锰处理24小时TUNEL染色阳性细胞随着氯化锰浓度的增加而增多;Cleaved caspase-3染色阳性细胞亦随着氯化锰浓度的增加而增多。结论通过大鼠耳蜗器官体外培养方法,发现氯化锰会造成毛细胞缺失,神经纤维变细变少,而这种损伤的机制与细胞凋亡密切相关。

To study relations between manganese toxicity the damage and apoptosis of cochlear hair cells. Methods Cochlear organotypic cultures were prepared using postnatal day 3 Sprague–Dawley rats. Mn chloride stock solution was prepared at a stock concentration of 100 m M in serum-free medium and diluted to final concentrations of 1, 3and 5 m M. Cochlear basilar membrane explants were incubated in the presence of the prepared Mn chloride solution. Stereocilia bundles of hair cells were immunolabeled with FITC phalloidin while nerve fibers were labeled with an antibody against the β–tubulin so that hair cells and nerve fibers in the cochlear basilar membrane were respectively observed under a fluorescent micro-scope. TUNEL kit and Cleaved caspase-3 antibody were used to label apoptosis of basilar membrane cells. Results Cochlear organotypic cultures treated for 24 h with 1m M Mn showed no obvious damage to the hair cells and auditory nerve fibers in the cochlear basilar membrane compared to the control group（P〉0.05）. Mn treatment at5 m M for 24 h caused noticeable damage to nerve fibers compared to the control（P〈0.05）. Damage by Mn treatment at 2m M was more obvious in the basal turn than in the middle and apical turns. At 24 h of Mn treatment, the number of TUNEL（as well as cleaved caspase-3） labeling cells increased along with increasing Mn concentrations. Conclusion In this in vitro organotypic culture of rat cochlea, Mn can cause missing of hair cells and reduced number and size of nerve fibers, which may be related to the mechanisms of cellular apoptosis.