短时培养过程中单离耳蜗外毛细胞的变性

Degeneration of isolated cochlear outer hair cells in short-term culture

目的观察体外短时培养中单离外毛细胞的变性过程,探讨单离毛细胞体外培养应解决的问题。方法应用酶消化结合机械吹打分离14天龄小鼠耳蜗毛细胞,在倒置相差显微镜下,观察和记录单离外毛细胞体外变性的形态变化。结果二侧耳蜗平均可获得46±19个活性良好的外毛细胞。在短时体外培养中部分外毛细胞活性保持良好。细胞变性主要表现为细胞水肿、变形,胞浆内出现是布朗运动样颗粒及胞膜破裂后胞浆内容物向细胞外溢出等现象。结论体外培养过程中产生毛细胞变性可能是由于毛细胞分离过程中的机械损伤和细胞能量供给不足所致。

PURPOSE To observe outer hair cells(OHCs)supply.degeneration in short-term culture and discuss the remaining problems in isolated hair cells culture.METHODS OHCs were isolated from 14-day-old mouse cochlea with incubation of the organ of Corti with papain(0.25mg/ml,10min,at 37℃)and trituration by a pipette.Morphological features of the degenerative isola ted OHCs were observed under the reversed phase contrast microscope(NIKON)during short-term cultrue in vitro.RESULTS there were 46±19 healthy OHCs obtained from two cochleae averagely.Isolated OHCs could be in good condition in culture for several hours.Typical signs of degeneration were swelling and distortion of the isolated OHCs,increased Brownian motion in the cytoplasm,and extravasation of the cytoplasmic organelles in Brownian motion from leaking along the damaged membrane.CONCLUSIONS The possible causes of OHCs degeneration in short-term culture may be mechanical dama ge in preparing isolated OHCs and the shortage of energy