GJB2基因条件敲除小鼠耳蜗外毛细胞凋亡时程观察

Apoptosis Process of Outer Hair Cells in Organ of Corti in GJB2 Conditional Knock out Mice Cochlea

目的探讨GJB2基因条件敲除小鼠耳蜗毛细胞缺失方式和具体时间。方法 GJB2基因条件敲除小鼠cCx26loxp/loxp-Pax2-Cre(cCx26)由转基因小鼠Cx26loxp/loxp与Cx26loxp/--Pax2-Cre小鼠杂交获得,分别选取P8、P14、P18和P21四个发育阶段各6只小鼠进行实验。野生型BALB/c小鼠作为正常对照。常规解剖出耳蜗基底膜,鬼笔环肽-FITC及碘化丙啶(PI)分别染色、铺片,激光共聚焦显微镜观察、拍照。结果与野生型小鼠相比,cCx26小鼠P8时耳蜗外毛细胞纤毛、细胞核形态均未见明显异常改变;P14时,鬼笔环肽染色显示耳蜗中回表皮板疏松,外毛细胞纤毛染色开始不规则,PI染色外毛细胞核排列极性欠佳,细胞核着色不均匀,可见深染的胞核,尚未见明显的外毛细胞缺失;P18时,鬼笔环肽染色显示表皮板出现区域性的模糊和单个外毛细胞纤毛的消失,外毛细胞核出现皱缩、片断化,甚至缺失,这一改变以中回最为明显,底回次之,顶回改变最轻微;P21时,表皮板出现了片状缺失,外毛细胞丢失明显,残余细胞排列紊乱。与野生型小鼠相比,cCx26ko小鼠各阶段内毛细胞纤毛染色不规则,深浅不一,细胞核未见明显形态异常及缺失。结论 GJB2基因缺失可引起小鼠耳蜗Corti器表皮板破损以及外毛细胞的凋亡,表皮板及外毛细胞纤毛的变化开始于P14,P18时外毛细胞出现典型的凋亡表现。

Objective To study the death mode and accurate time point of outer hair cells in organ of Corti＇s of GJB2 conditional knock out mice cochlea. Methods Cochlea from P8, P14, P18 and P21 of cCx26ko（Cx26^loxp/loxp-Pax2--Cre） and wild type ones （BALB/c） were harvested. Basilar membranes were dissected out and stained by fluorescein isothiocyanate （FITC）--conjugated phalloidin and propidium iodide （PI）. The surface morphology of the basilar membrane was observed under confocal microscope. Results There were no evident changes in the surface morphology of cochlea between knock out mice and wild type one in P8. Very slight changes, like loose cuticular plate and dark stained OHCs nuclei appeared in P14. Obvious changes in surface morphology of cCx26ko mice coch- lea were observed at P18 stage. The OHC nuclei were shrunken, condensed, fragmented, and in some cases, com- pletely missing sporadically. The FITC phalloidin labeling of the cuticular plate around the OHCs appeared fuzzy and irregular, which may indicate cuticular plate damage. In P21, the damage was much more evident. Breakage and segmental deletion of basilar membrane and disarrangement of hair cells nuclei were found. The aforementioned changes were more severe in the middle turn in the basal turn and the apical one. Oorlclusion The deletion of GJB2 gene may cause damage of cuticular plate and apoptosis of outer hair cells in mice cochlea. The damage started from P14 and evident apoptosis of outer hair cells was revealed in P18.