RA诱导P19细胞神经分化过程中相关蛋白分子的表达变化

Related proteins expressional change during retinoic acid-induced neural differentiation of P19 cells

目的:研究维甲酸(retinoic acid,RA)诱导P19细胞神经分化过程中与增殖、分化及凋亡相关分子表达变化情况。方法:P19细胞经RA处理4 d后进行分化培养,对分化培养细胞用MAP2抗体进行免疫组织化学染色。在P19细胞分化前、后不同时间点,Western Blot检测细胞周期蛋白(Cyclin D、Cyclin E、Cyclin A、p-CDK2)、生长分化相关蛋白(E2F1、PCNA、GAP43)、MAGE家族蛋白(Necdin、MAGE D1、MAGE H1)及凋亡效应分子(激活型Caspase 3)表达变化情况。结果:RA可诱导P19细胞向神经元分化;Necdin、GAP43和MAGE D1在RA处理后明显上调并在分化过程中保持高水平表达。E2F1、Cyclin D和Cyclin E表达随分化进程呈现逐步升高趋势,而Cyclin A、p-CDK2表达逐步下调。MAGE H1在分化第4 d到达顶峰后下降。PCNA在RA处理4 d时表达下降至最低,在分化早期渐上调后又降至低水平。RA处理后激活型Caspase 3表达明显上调,但在随后分化过程中又下调至对照组水平。结论:RA诱导P19细胞向神经元分化过程中,与细胞生长、分化、增殖及凋亡相关蛋白呈现动态表达变化,为进一步研究这些分子在神经分化中的作用奠定了基础。

Objective： To explore the expression of proteins related with proliferation,differentiation and apoptosis during retinoic acid（RA）-induced neural differentiation of P19 cells.Methods： P19 cells were differentially cultured after RA-treated for 4 days.The differentiated cells were identified through immunohistochemical staining with antibodies against MAP2.At different time points before or after differentiation,Western Blot was done to detect the expressional levels of the proteins,including cell cycle proteins（Cyclin D,Cyclin E,Cyclin A,p-CDK2）,growth and differentiation-related proteins（E2F1,PCNA,GAP43）,mage family proteins（Necdin,MAGE D1,MAGE H1） and active Caspase 3.Results： RA treatment could induce the differentiation of P19 cells to neurons.After RA induction,the expression of Necdin,GAP43 and MAGE D1 in P19 cells were significantly increased and sustained such levels during subsequent differentiation.With the differentiation of P19 cells,the expressional levels of E2F1,Cyclin D and Cyclin E were gradually ascend,but Cyclin A and p-CDK2 descend.The expressional level of MAGE H1 reached to its peak at 4 days after differentiation and then decreased.For PCNA,its expression reached rock-bottom at 4 days after RA treatment,followed by temporarily increase at the early differentiation.RA could increase the expression of active Caspase 3,which dropped to control levels with differentiation.Conclusion： During the RA induced neural differentiation of P19 cells,the expression of growth,differentiation,proliferation and apoptosis-related proteins were dynamically presented,all of which might lay a foundation for further exploring the roles of those molecules in neural differentiation.