Yuahasi Katia K, Demasi Marcos A, Tamajusuku Alessandra S K, Lenz Guido, Sogayar Mari C, Fornazari Maynara, Lameu Claudiana, Nascimento Isis C, Glaser Talita, Schwindt Telma T, Negraes Priscilla D, Ulrich Henning
Departamento de Neurologia Clínica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Int J Dev Neurosci. 2012 Apr;30(2):91-7. doi: 10.1016/j.ijdevneu.2011.12.010. Epub 2012 Jan 13.
Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models.
胚胎癌细胞是研究早期胚胎发育过程中增殖和分化机制的广泛应用的模型。我们现在研究了通过RNA干扰（RNAi）下调P2X2和P2X7受体表达如何影响P19胚胎癌细胞的神经分化和表型特化。野生型P19胚胎癌细胞或稳定表达靶向P2X2或P2X7受体表达的短发夹RNA（shRNA）的细胞在视黄酸存在下被诱导分化为神经元和神经胶质细胞。在分化过程中沉默P2X2受体表达促进了细胞增殖，并增加了表达神经胶质特异性GFAP的细胞百分比，同时β-3微管蛋白阳性细胞的数量减少。在稳定的抗P2X2受体RNAi存在下诱导增殖表明了一种机制，即神经胶质细胞增殖优于祖细胞的生长停滞，而祖细胞的生长停滞会允许神经元成熟。与P2X2受体不同，在P19细胞神经分化过程中抑制P2X7受体表达导致细胞增殖和GFAP表达减少，这表明神经胶质生成的进展需要功能性P2X7受体。本研究获得的结果表明嘌呤能信号在神经分化过程中细胞命运决定中的重要性，P2X2和P2X7受体分别促进神经发生和神经胶质生成。在这项工作中开发的下调P2X2或P2X7受体基因表达的shRNA，为研究其他干细胞模型中的神经分化机制提供了有用的工具。
