小鼠胚胎干细胞Notch1基因沉默对糖原合酶激酶3β和周期蛋白依赖性激酶5表达的影响

Effects of Notch1 gene silencing in mouse embryonic stem cells on glycogen synthase kinase-3 beta and cyclin-dependent kinase 5 expression

背景:GSK-3β与CDK-5都属于磷酸化蛋白激酶,一并参与胚胎神经细胞分化发育具体机制尚不清楚。目的:观察胚胎干细胞Notch1基因沉默后Hes-1、PS1、Gsk-3β和Cdk5表达的变化以及Notch信号对Gsk-3β和Cdk5表达的调节作用及对早期神经细胞分化发育的潜在影响。方法:通过阳离子脂质体转染法将Notch1siRNA真核表达载体转入小鼠胚胎干细胞。倒置显微镜下观察转染前后细胞的形态变化;48h后收集细胞,RT-PCR检测包括Notch1在内的Hes-1、PS1、Gsk-3β和Cdk5基因水平表达情况,单因素方差分析各组间差异。结果与结论:①RT-PCR检测到转染pSINsi-U6-Notch1质粒组胚胎干细胞Notch1、Hes-1、GSK-3β和Cdk5的表达情况较转染空质粒组与正常对照组显著降低(P<0.05),转染空质粒组与正常对照组之间无明显差异(P>0.05,n=5)。②倒置显微镜下观察未见外源质粒的转入对胚胎干细胞克隆性生长的影响,只是转染pSINsi-U6-Notch1质粒组胚胎干细胞的增殖量较其他两组有所降低。③胚胎干细胞PS1表达情况3组间无显著差异(P>0.05)。可以看出,胚胎干细胞Notch1基因表达水平的降低导致蛋白激酶GSK-3β、Cdk5表达的下调。

BACKGROUND：Glycogen synthase kinase-3β（Gsk-3β） and cyclin-dependent kinase 5（Cdk5） are phosphorylated protein kinase,and participate in differentiation and development of embryonic neuronal cells,but the precise mechanism remains unclear.OBJECTIVE：To detect the changes in genetic expression of Hes-1,PS1,Gsk-3β and Cdk5 after Notch1 gene silencing in embryonic stem cells（ESCs）,thus to explore Notch1 signaling accommodative action on Gsk-3β and Cdk5 and its potential impact on early neurogenesis.METHODS：Lipofectamine 2000 was used to transfer Notch1 small interfering RNA（siRNA） eukaryotic expression vector into mouse ESCs.Morphologic changes of cells before and after transfection were observed under inverted microscope.ESCs were collected at 48 hours after transfection.Genetic expressions of Notch1,Hes-1,PS1,Gsk-3β and Cdk5 in these cells are detected by RT-PCR.The intergroup differences were analyzed using one-way variance analysis.RESULTS AND CONCLUSION：① Relative expression of Notch1,Hes-1,Gsk-3β and Cdk5 in pSINsi-U6-Notch1 group was obviously lower than blank plasmid and normal control groups（P 0.05）.There were no significant differences between blank plasmid and normal control groups（P 0.05,n=5）.② Under an inverted microscope,exogenous plasmid did not impact ESCs cloning growth,but the proliferation quantity of cells in pSINsi-U6-Notch1 group was decreased compared with blank plasmid and normal control groups.③ No significant differences in genetic expression of PS1 were detected among the three groups（P 0.05）.Decreased Notch1 gene expression in ESCs down-regulates gene expression of Gsk-3β and Cdk5.