哺乳动物内耳再生毛细胞前体细胞来源研究

Origination of progenitor cells of regenerative hair cells in rats

目的探讨哺乳动物内耳再生毛细胞前体细胞的可能来源。方法取出生1d大鼠的椭圆囊，嗜热菌蛋白酶处理，消化法原代培养。在倒置显微镜下，对椭圆囊感觉上皮细胞（USEC）的形态、生长特征进行观察；透射电镜及免疫细胞化学法检测细胞角蛋白18、波形蛋白；免疫细胞化学法及RT—PCR检测支持细胞标记物p27kipl mRNA及毛细胞的特征性标记物Brn3a、Calretinin及AchRa9、Myosin Vlla mRNA的表达，进而鉴定USEC上皮细胞来源。结果原代培养的USEC呈扁平、多角形、核大而圆的上皮细胞形态，细胞问连接紧密，形成单层时呈“铺路石样”外观，可见“dome”结构；表达细胞角蛋白，但不表达波形蛋白，细胞微绒毛丰富，细胞问连接紧密。原代培养的USEC表达支持细胞标记物p27kipl mRNA及毛细胞的特征性标志物Brn3a、Calretinin及AchRa9、Myosin Vlla mRNA。结论原代培养的USEC可以产生毛细胞样细胞，而且能表达支持细胞标记物，表明其来源为支持细胞。椭圆囊感觉上皮的支持细胞可能是毛细胞再生的前体细胞之一。

Objective To investigate the origin of progenitor cells of regenerative hair cell in mammalian. Methods Utricular sensory epithelial from postnatal dl Wister rats was isolated by mechanical dissociation. The explants were digested by thermolysin, then transferred to an aliquot containing trypsin and collagenase for incubation, and the pure utricular sensory epithelial cells （USECs） were harvested. USECs were cultured in primary culture, and observed with inverted microscope. Cytokeratin 18 and vimentin were detected by immunocytochemical method; Brn3a and calretinin were detected by immunofluorescence method; cell ultrastructure was examined with transmission electron microscope; mRNA of p27kipl, AchRa9 and Myosin Vlla was measured with reverse transcription PCR. The markers of hair cells and supporting cells were used to identify the origination and characterization of USEC. Results USECs presented a large, flat, polygonal epithelial morphotype with big, round nuclei. USEC grew into monolayer with ＂ cobblestone- like ＂ appearance. Some cells presented ＂Dome＂ formation, probably due to fluid collection underneath the cell monolayer. The culture USECs expressed cytokeratin 18, but no vimentin, had rich microvilli and complex tight junction, which indicated the epithelial origination of USEC. The cells expressed marker of supporting cell （p27kipl mRNA）, and characteristic markers of the hair cells （Brn3.a, Calretinin and AchR a9, Myosin Vlla mRNA）. Conclusion USECs co-expressing the characteristic markers of the hair cells and supporting cells, which indicated that USECs can be the origin of the regenerated hair cells and the progenitor cell may come from supporting cells.