摘要
目的探讨维甲酸(RA)诱导胚胎干细胞(ESC)向神经细胞定向分化及自由分化过程中,Notch1蛋白和mRNA的时空表达以及RA对Notch1表达的影响。方法实验分两组:实验组用RA对ESC进行神经细胞定向诱导分化,对照组ESC进行自由分化。各组分别取ESC分化1、3、5、7、9d的细胞,倒置显微镜观察形态变化,免疫荧光观察MAP2表达,免疫细胞化学、流式细胞术及RTPCR检测Notch1蛋白和mRNA表达。结果实验组诱导分化的神经元逐渐增多并形成神经网络。对照组以圆形上皮样分化细胞为主。ESCNotch1蛋白及mRNA均为高表达,诱导分化或自由分化后蛋白表达降低(P<0.05)。实验组Notch1mRNA水平下降(P<0.05),对照组则无明显变化(P>0.05)。分化3d后,实验组各时间点Notch1的表达水平均低于对照组(P<0.05)。结论ESC分化时伴有Notch1信号的关闭。Notch1可能对ESC向神经元的特异性分化起重要作用。促进Notch1的下调可能是RA诱导分化的机制之一。
ObjectiveTo investigate the expression of Notch1 protein and mRNA during retinoid acid (RA) induced differentiation of embryonic stem cells(ESC) and the effect of RA on Notch1 expression.MethodsESC were committedly induced to neural cells by RA in experimental group and randomly differentiated in a control group.ESC and differentiated cells were collected at 0 (ESC),1,3,5,7 and 9 days in each group for the morphologic observation under the inverted microscope.MAP-2 antigen was detected by immunofluorescence cytochemical method.Notch1 protein and mRNA expressions were determined by immunocytochemistry,flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR).ResultsRA-induced neural cells were significantly increased in comparison with control group and showed a neural network structure in experimental group and epithelial-like cells in the control group.Notch1 protein and mRNA levels reached a peak in ESC phase and Notch1 protein was gradually decreased after induced or random differentiation(P<0.05).Notch1 mRNA showed a reduced expression in experimental group(P<0.05) but not in control group(P>0.05).Notch1 protein and mRNA levels in experimental group were lower than those in control group after differentiation for 3 days(P<0.05).ConclusionNotch1 signal is closed during ESC differentiation.It may play an important role in maintaining ESC’s stem cell characteristics and is essential for the committed neural cell differentiation.RA promotes the downregulation of Notch1 expression,suggesting its interaction with Notch1 factor is probably one of the RA induction mechanisms.
出处
《眼科研究》
CSCD
北大核心
2005年第3期241-244,共4页
Chinese Ophthalmic Research
基金
国家杰出青年基金(30125041)
教育部博士点专项科研基金(A032003149)
广东省教育厅"千百十工程优秀人才"培养基金(Q02027)
中国博士后科学基金(2004036520)资助
