| Summary: | 碩士 === 高雄醫學大學 === 生理及分子醫學研究所碩士班 === 96 === The differentiation of mammalian neurons during development is a highly complex process involving regulation and coordination of gene expression at multiple steps. In immature neurons, [Cl−]i is high. The activation of GABA- or glycine-gated Cl− conductances results in membrane depolarization. This lead to activation of voltage-dependent Ca2+ channels (VDCCs) and [Ca2+]i transients that have a central role in neuronal differentiation, growth and maturation. In addition, intracellular pH (pHi) regulates many processes important for cell growth and differentiation.
Cl-/HCO3- exchanger (AE) activity is necessary for maintaining pHi in embryonic cells. AE is also responsible for Cl- influx. However, the role of Cl- transporter during neuronal differentiation is not clear. We examined the effect of Cl- transport inhibitor 4,4''-diisothiocyana-
tostilbene-2,2''-disulphonic acid (DIDS) on the neuronal differentiation of P19 cells. We used class III β tubulin as the differentiated neuron marker and Oct-3/4 as the undifferentiation stem cell marker. In comparison with RA (500nM) only treatment, combined treatment of RA (500nM) and DIDS (200μM) significantly increased the expression of class III β tubulin while decreased the expression of Oct-3/4, revealed by immunoblotting, real-time PCR and immunofluorochemistry. Another Cl- channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) was also tested. Our experiment showed that combined treatment of RA (500nM) and NPPB (50μM) significantly increased the expression of class III β tubulin. We further examined the effect of changing pHo during the neuronal differentiation of P19 cells. We found that treatment of RA in low pHo significantly increased the expression of class III β tubulin. P19 cells express Cl-/HCO3- exchanger isoform protein AE3. Our results suggest that the inhibition of Cl- channel and the stimulation of AE3 may cause Cl- accumulation in P19 cells, leading to enhancement of RA-induced P19 neuron differentiation.
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