Differentiation Of Cochlear Neural Progenitor Cells Induced By Retinoic Acid

Objective: To study the differentiation of cochlear neuralprogenitor cells into inner ear hair cells induced by retinoic acid.Methods: After thawing,a pipe of cochlear neural progenitor cells(CNPs) is cultured in a serum-free medium (containing epidermal growthfactor、basic fibroblast and N2supplement).3days later,growing cellsin well condition was observed under an inverted microscope,then wasidentified by Nestin antibody.The experiment was divided in the experimental group and control group.Cochlear neural progenitor cells were differentiated by Medium which waschanged every day.3days later cells were collected for furtherexperiment. Experimental groups were treated as follows:①Cochlearneural progenitor cells were induced differentiation in DMEM/F12+RA (10-6M)medium.②Using immunofluorescence to detect hair cell markers-Math1,Myosin Ⅶ.③Extracting RNA of the induced cells to detecte integrityby agarose gel electrophoresis; Using RT-PCR to detect the expression ofthe hair cell marker gene;④Cell proliferation was detected by Brdufluorescence stainings.The expressions of F-actin were detectedd byphalloidin fluorescence stainings.The control group cells were inducedby DMEM/F12+DMSO (0.11mg/ml).Results:1.The expression of nestin can be seen in Cochlear neural progenitorcells.2.Cell immunofluorescence showed that the experimental group and thecontrol group were visible fluorescence, The experimental group and thecontrol group Math1+, Myosin Ⅶa+cell rates were63%,1.5%,56%,0.96%. 3.RNA electrophoresis showed three clear bands.4.RT-PCR electrophoresis results showed that hair cell marker geneexpressed in both experimental group and control group, butElectrophoresis bands of experiment group were more bright.5.Cell proliferation assay showed that Brdu+cell rate in RA group(20.6%) is less than the negative control group (29.9%) and positivecontrol group (54.3%), negative control group and positive control groupwere also different.6.F-actin fiber of Control group directed inconsistently.Thefluorescence intensity of Ctrl group、 the control group、 theexperimental group showed no difference,the figures were21.60,22.60,24.50.Conclusions:1.The cochlear neural progenitor cells (CNPs) in serum-free conditionswere in undifferentiated condition, still maintained the characteristicsof neural stem cells.2.RA can promote cochlear neural progenitor cells into the inner earhair cells.3.RA inhibits in vitro proliferation of cochlear neural progenitorcells; RA promote F-actin skeleton ear reconstruction, but did notpromote the formation of F-actin cytoskeleton.