Hath1基因转染后大鼠耳蜗毛细胞前体细胞的扫描电镜观察

目的探讨Hath1基因对大鼠耳蜗大上皮嵴(greaterepithelialridge,GER)细胞的诱导分化作用。方法取出生后第1天的大鼠耳蜗,利用机械分离和酶消化相结合的方法分离出纯的GER细胞,于含10%血清DMEM培基中进行培养。以腺病毒为载体对GER培养物进行Hath1基因的转染,并对感染后10天的标本进行扫描电镜观察。结果扫描电镜观察显示GER细胞在含10%血清DMEM培基中呈片状贴壁生长特性,表现为典型的上皮样多角型外观。Hath1感染后10天的GER细胞培养物可见在细胞团的边缘出现了细丝状纤毛样结构,而无病毒感染对照组未见到该结构。结论GER细胞作为毛细胞前体细胞可以实现体外原代培养,在Hath1基因重表达情况下,部分细胞的表面可见不成熟的纤毛样结构,提示Hath1基因也许可以诱导离体培养下的纯的毛细胞前体细胞向毛细胞的初步分化。

SEM observation of hair cell progenitors forced to express Hath1 from rat cochleae

OBJECTIVE To investigate the effect of Hath1 on epithelial ridge (GER) cells from postnatal rat cochlear. METHODS An experimental method was developed which allowed the isolation and culture of GER cells from P1 rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation. The dissociated GER cells were cultured in DMEM 10% FBS. The GER explants infected with ad-Hath1 for 3 hours were cultured for 10 days and observed under scanning electron microscope. RESULTS The GER cell cultures tended to attach to the substratum and grow in patches which assume a polygonal morphology similar to that of epithelial cells in medium containing 10% serum. Stereociliary bundle-like structures were observed in the boundary of GER cell patches forced to express Hath1. CONCLUSION That GER cells which are likely hair cell progenitors could be cultured in vitro and generate stereociliary bundle-like structure when forced to express Hath1 suggests that the misexpression of Hath1 probably can induce the predifferentiation of pure hair cell progenitors into hair cells in vitro.