蛋白激酶Cα在小鼠胚胎干细胞诱导分化为神经样细胞中的表达变化

目的研究小鼠胚胎干细胞(embryonic stem cells,ESC)定向诱导分化为神经样细胞过程中蛋白激酶Cα(protein kinase Cα,PKCα)的表达变化,探讨可能涉及ESC定向分化的信号机制.方法ES-D3细胞株用不含小鼠白血病抑制因子(mouseleukemiainhibitoryfactor,mLIF)的ESC培养液培养4 d,形成胚胎体(embryoid bodies,Ebs),再分别种植于预置有盖玻片的6孔板和直径100mm的培养皿,经5×10-7 mol/L维甲酸(retinoic acid,RA)诱导后,用神经特异性烯醇化酶(NSE)和NF-200来鉴定分化细胞的类型,在诱导后第1、3、5、7和14天用Western印迹和逆转录聚合酶链反应(RT-PCR)方法检测PKCα亚型的表达变化.结果诱导前免疫组化发现PKCα在ES-D3细胞有广泛的棕黄色的阳性表达,以细胞浆和细胞膜最明显;Western印迹发现PKCα出现一条蛋白印迹条带,分子量约为84kD;诱导后第3天,NSE和NF-200开始表达,第7天达到高峰;诱导后Western印迹和RT-PCR方法都显示PKCα表达量急剧下降,以后逐渐升高,至第14天基本恢复至正常水平.结论在RA诱导ESC定向分化为神经样细胞过程中,PKCα有独特的时空表达特点,它可能对ESC的分化有非常重要的调控作用.

Expression Changes of Protein Kinase Cα during Differentiation of Mouse Embryonic Stem Cells into Neuron-like Cells

PURPOSE: To study the expression of protein kinase Calpha (PKCalpha) during differentiation of mouse embryonic stem cells (ESC)into neuron-like cells in an attempt to elucidate their role in signaling. METHOD: ES-D3 cells were subjected to an 18-day induction procedure which consisted of 4 days of culture as embryoid bodies (EBs) without all-trans retinoic acid (RA) followed by 14 days of culture in the presence of RA, then the EBs were plated separately onto gelatin-coated 6-well culture dishes coated glass coverslip for immunohistochemistry, onto 100-mm-diameter bacteriological (nontissue culture) dish for Western blot assay and RT-PCR, and were cultivated and collected cells for 1, 3, 5, 7 and 14 days in the presence of RA. RESULTS: Staining of PKCalpha was strong and distributed throughout the cells, especially in the cytoplasm and membrane. Western blot analysis exhibited a band of approximately 84 kD with the antibody specific to PKCalpha. After induction of RA, PKCalpha was present in significantly lower levers in induced ESC as compared with that in ES-D3 cells at first, then PKCalpha isoform was found in slightly higher amounts and resumed on 14th day. CONCLUSION: This study was characterized the PKCalpha isoenzyme profile in RA-induced differentiation of ESC into neuron-like cells and suggests that PKCalpha may play a very important role in differentiation of mouse ESC along the neuronal pathway.