Long-term culture of utricular sensory epithelial cell of rats and expression of the hair cell characteristic markers / 临床耳鼻咽喉头颈外科杂志

ABSTRACT

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 Ⅶa.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 neuclei.The cells grew into monolayer,cobblestone-likeappearance 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 Ⅶa 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.