大鼠椭圆囊感觉上皮细胞长期培养体系的建立及毛细胞标记物表达的意义

Long-term culture of utricular sensory epithelial cell of rats and expression of the hair cell characteristic markers

目的:建立椭圆囊感觉上皮细胞(USEC)长期培养体系,为內耳毛细胞再生研究提供稳定的活性细胞。方法:取1d龄wistar大鼠,在显微镜下取出椭圆囊上皮,嗜热菌蛋白酶处理,获得具活性的纯椭圆囊感觉上皮,胰蛋白酶+胶原酶消化,培养传代,传25代。取第25代USEC在倒置显微镜、透射电镜下对USEC的生长特征、形态结构进行观察;免疫细胞化学检测细胞角蛋白18、波形蛋白、Brn3.a及Calretinin的表达。RT-PCR检测毛细胞的特征性标记物AchRa9、MyosinⅦamRNA的表达。结果:USEC细胞培养达6个月传25代,第25代USEC均呈扁平、多角形、核大而圆的上皮细胞形态,细胞之间连接紧密,形成单层时均呈"铺路石样"外观,可见dome结构。该细胞表达角蛋白18和不表达波形蛋白,微绒毛丰富,细胞间连接紧密,提示其上皮起源。第25代USEC表达毛细胞的特征性标志物Brn3.a、Calretinin及AchRa9、MyosinⅦamRNA,表明长期培养USEC仍具有毛细胞的前体细胞的特性。结论:本实验成功建立了USEC长期培养体系。长期培养的USEC性状未发生明显改变,表达上皮细胞及毛细胞的特征性标志物,具有毛细胞前体细胞的特征。这为毛细胞再生的机制及内耳分子、基因研究提供了稳定的细胞来源。

Objective：To investigate the generative or regenerative hair cell and studes of molecular and genetic in the inner ear, the long-term culture systems of utricular sensory epithelial cell of the rats were established. Method：Utricular sensory epithelial of postnatal day 1 rats was isolated by mechanical dissociation. The explants were digested by thermolysin, then transfrred to an aliquot containing trypsin and collagenase for incubation to harvest the pure utricular sensory epithelial cell. USEC wes cultured in Dulbecco modified Eagle medium（DMEM） and passaged. USEC of the 25 passages observed by inverted microscope and ultrastrctural examination with transmission electron microscope. Immunocytochemical method with cytokeratin 18 , vimentin,Brn3, a and Calretinin; reverse transcription PCR with mRNA of AchRa9 and Myosin Ⅶ a. The markers of hair cells were used identify the characteriztion of USEC of the 25 passages. Result： USEC have been cultured for more than 6 months and passaged 25 passages. USEC of the 25 passages showed a large, flat, polygonal epichlial morphotype with big, round neuciei. The cells grew into monolayer, ＂cobblestone-like＂appearance and showed ＂Dome＂ formation. The cells expressed cytokeratin 18 , not expressed vimentin, has rich microvilli and complex tight junction,which indicated the epithlial origination of USEC. Coexpressed of the hair cell characteristic markers Brn3. a,Calretinin and mRNA of AchRa9, Myosin Ha was identified the culture cell that may represent rat progenitor hair cell. Conclusion： The long-term culture systems of utricular sensory epithelial cell of the rats were successfully established. The long-term culture USEC coexpressed the characteristic markers of the hair cell ,which identified the culture cell may represent progenitor cell of rats hair cell . It may provide valuable cell sources for in-depth investigation the mechanisms of hair cell generation or regeneration and studes of molecular and genetic in the inner ear.