豚鼠耳蜗Hensen细胞的分离、活性鉴定及其胞内静态游离Ca~(2+)分布

使用酶消化法及机械分离法对 Hensen细胞进行分离 ,置于倒置显微镜下用碘化丙啶及钙敏荧光染料 Fluo-3进行细胞活性鉴定并在激光扫描共聚焦显微镜下观察静息状态 Hensen细胞内的游离 Ca2 +的时空分布。结果证明 ,每个豚鼠耳蜗可以得到单离的 Hensen细胞 5～ 12个。细胞活性良好 ,可保持 5～ 6h。当细胞变性、坏死时可见一系列的形态学变化。对此得出几点判断 Hensen细胞活性的标准 :(1)细胞体呈卵圆形或椭圆形 ,大小可不一致 ;(2 )细胞膜完整 ,细胞边界清楚 ,折光现象明显 ;(3 )细胞浆清澈透明 ,无布朗运动 ,无溢出 ;(4 )细胞浆内的脂滴清晰可辨 ,折光现象明显 ;(5 )细胞无水肿 (即无气球样变 )。在静息状态下 ,Hensen细胞内的 Ca2 + 在细胞内分布不均匀 ,脂滴所在部位没有 Ca2 + 的分布。随时间延长 ,细胞内的 [Ca2 + ] i可小幅度振荡 ,但基本处于一种稳定状态。本工作为进一步对 Hensen细胞的其他特性进行研究打下了基础

ISOLATION, VIABILITY ASSESSMENT AND STATIC FREE Ca2+ DISTRIBUTION IN HENSEN CELL OF THE GUINEA PIG COCHLEA

Enzymatic and mechanical method was used to isolate Hensen's cell. The cell's viability was judged with propidium iodide and Fluo 3 under inverted microscope and then Ca 2+ distribution was observed in the cell with laser scan confocal microscope. We could get 5 to 12 Hensen's cells with good viability from each guinea pig cochlea. The viability could last 5 to 6 hours. There were a series of morphological changes when the cells underwent degeneration and necrosis. Some standards ccold be used for assessing the viability of Hensen's cell: (1) the shape of the cell body appeared to be oval round or ellipse and its boundary was clear; (2) the cell's membrane was intact with apparent refraction and without cytoplasm leaking out; (3) the fat droplets in the cytoplasm were conspicuous with apparent refraction. (4) there were small granulates in the cell distributed evenly in the cytoplasm, which might be organells. Under static situation, the Ca 2+ concentration was in a gradient from center to edge, high in the center and low on the edge. The [Ca 2+ ]i in Hensen's cell was in homeostatic state. This work set up a foundation for further study on the characteristic of Hensen's cell.