耳蜗毛细胞凋亡早期的检测方法

The method of detecting the apoptotic hair cells at their early stage in the cochlea

  • 摘要: 目的: 介绍一种耳蜗毛细胞凋亡早期的检测方法。方法: 将豚鼠暴露于155dBSPL的脉冲噪声75次,于噪声暴露后5min解剖取耳蜗。采用异硫氰酸荧光素标记的鬼笔环肽(FITC-labeledphalloidin)进行毛细胞表皮板和纤毛肌动蛋白的染色,DNA荧光染料碘化丙锭(propidiumlodide, PI)进行细胞核染色,标记鉴别毛细胞死亡方式。原位末端标记法(TUNEL)进一步确认凋亡的毛细胞,荧光显微镜下观察凋亡的毛细胞形态学变化。结果: 异硫氰酸荧光素标记的鬼笔环肽和PI双重染色发现毛细胞核发生轻微固缩时,其表皮板结构完好。TUNEL阳性标记物存在于固缩的细胞核内。结论: 应用异硫氰酸荧光素标记的鬼笔环肽和PI双重染色法能够发现凋亡早期的毛细胞。

     

    Abstract: Objective:To introduce a method of detecting the apoptotic hair cells at their early state in the cochlea. Methods:Guinea pigs were exposed to 75 pairs of impulse noise (1 second interval between each pair) at 155 dB pSPL. 5 min after the noise exposure, the organs of corti were processed for detection of hair cell death modes. The apoptotic hair cells were distinguished by double-stained using FITC-labeled phalloidin for filamentous actin (F-actin),propidium iodide (PI) for hair cell nuclei and confirmed by staining for TUNEL assay. The specimens were examined under a fluorescence microscope for assessment of the hair cell damage. Results:Some slightly condensed nuclei can maintain the integrity of F-actin staining in the tissue double-stained with FITC-labeled phalloidin and PI. In TUNEL labeled cochlea, there is strong green fluorescence in the condensed nuclei. Conclusion:The method of double-stained with FITC-labeled phalloidin and PI can be used to distinguish the apoptotic hair cells at their early state.

     

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