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

OBJECTIVE

To investigate the generative or regenerative hair cell and studies 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 transferred 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 ultrastructural 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 characterization 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 epithelial morphotype with big,round nuclei. 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 epithelial 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 studies of molecular and genetic in the inner ear.