摘要
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 study the effect of protein kinase C_α on the expression of developmental genes pax-6, slit-2 and netrin-1 during differentiation of mouse embryonic stem cells into neuron-like cells in vitro, in an attempt to elucidate their roles in signaling.Methods ES-BALB/c cells were induced to form embryoid bodies in the ES conditioned medium for 4 days, and were plated separately on coated glass coverslip into 6-well culture dishes for immunohistochemical study and into 100 mm dishes for RT-PCR assay. These cultures were collected after 1, 3, 5, 7 and 14 days in the presence of 5×10^-7 mol/L retinoic acid (RA). The neuron-like cells were stained with antibody to NSE and NF-200. mRNA level of the development related genes (pax-6, slit-2 and netrin-1) in undifferentiated and differentiated ES cells was assessed by RT-PCR assay. Effects of PMA and D-sphingosine on the developmental genes were also observed.Results Most of the neuron-like cells stained with the antibodies to NSE and NF-200. RT-PCR assay showed that levels of PKC_α, pax-6 and netrin-1, but not slit-2 transcripts decreased dramatically upon induction on the 1st day, but then raised slowly and resumed to normal level on 14th day. PMA upregulated the levels of PKC_α, pax-6 and netrin-1; while D-sphingosine downregulated their levels. Conclusions The results of the present experiments demonstrate that the developmental genes pax-6 and netrin-1 play a very important role during differentiation of mouse ES cells into neuron-like cells through PKC_α signaling pathway.(Chin J Ophthalmol, 2005,41:123-127)
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2005年第2期123-127,共5页
Chinese Journal of Ophthalmology
基金
国家重点基础研究发展规划基金资助项目子项目(G1999054301)
国家高科技研究发展计划资助项目(2003AA205005)
国家自然科学基金资助项目(30171001
30200306)
广东省重点科技攻关资助项目(2003A3020401)
广东省科技攻关基金资助项目(2003C32715)
关键词
蛋白激酶CΑ
小鼠
胚胎干细胞
基因表达
调控
细胞分化
视神经疾病
Embryo
Stem cells
Protein kinase C
Cell differentiation
Gene expression regulation, developmental
Mice
