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目的探讨蛋白激酶Cα(PKCα)对小鼠胚胎干细胞(ESCs)定向诱导分化中发育基因(pax6、slit2、netrin1)表达的影响。方法ESBALB/c细胞株用无白血病抑制因子(mLIF)的ESCs培养液培养4d,形成胚胎体(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5×10-7mol/L视黄酸(RA)诱导后,用神经细胞特异性抗原NSE和NF200来鉴定分化细胞的类型,在诱导后1、3、5、7及14d用逆转录聚合酶链反应(RTPCR)方法测定细胞的PKCα和发育基因的mRNA表达情况,并观察了PKC激活剂佛波酯(PMA)和PKC抑制剂D鞘氨醇对它们表达的影响。结果免疫组化发现在诱导后第3d大多数细胞NSE和NF200染色阳性,RTPCR证实诱导后1dPKCα、pax6和netrin1的mRNA表达急剧下降,3~7d逐渐升高,至14d恢复至正常水平,而slit2在诱导前后均无明显表达。PMA可使PKCα、pax6、netrin1的mRNA水平在诱导后细胞中的表达明显增强,而D鞘氨醇的作用相反,使它们的表达显著降低,而对slit2无明显影响。结论发育基因pax6和netrin1在ESCs定向分化为神经样细胞中有重要的调控作用,可能是通过PKCα信号通路起作用。(中华眼科杂志,2005,41123127)
Objective To investigate the effect of protein kinase Cα (PKCα) on the expression of pax6, slit2 and netrin1 during mouse embryonic stem cells (ESCs) induced differentiation. Methods ESBALB / c cells were cultured in ESCs without leukemia inhibitory factor (mLIF) for 4 days to form embryos (EBs), which were then planted in 6-well plates with preset glass coverslips and 100 mm diameter dishes. After induction with 5 × 10-7mol / L retinoic acid (RA), NSE and NF200 were used to identify the type of differentiated cells, and 1, 3, 5, 7 and 14 days after induction with reverse transcriptase polymerase chain reaction (RTPCR) method was used to detect the mRNA expression of PKCα and developmental genes. The effects of PKC activator phorbol ester (PMA) and PKC inhibitor D-sphingosine on their expression were also observed. Results Immunohistochemistry showed that NSE and NF200 staining were positive on the third day after induction. The mRNA expression of PKCα, pax6 and netrin1 was decreased sharply on the 1st day after RTPCR induction, and gradually increased on the 3rd to 7th day and returned to the normal level on the 14th day. No significant expression was found before and after induction. PMA enhanced the expression of PKCα, pax6 and netrin1 mRNA in induced cells, but the effect of D-sphingosine was reversed, which significantly decreased their expression but had no significant effect on slit2. Conclusion The developmental genes pax6 and netrin1 play an important regulatory role in the directional differentiation of ESCs into neuron-like cells, probably through the PKCα signaling pathway. (Chinese Journal of Ophthalmology, 2005,41123127)