The Change of HES-1、PS-1 and GSK-3β’s Expression in Mouse ESCs after Transfection of Notch1
|Keywords||Embryonic stem cells Notch signaling pathway Wnt signaling pathway Crosstalk PS-1|
Background Notch signaling pathway is an evolutionarily very conservative signal transduction system. Developmental processes in invertebrates and vertebrates, Notch signaling plays a key role in the cell fate decisions. Notch receptor signaling can expand and solidify the molecular differences between adjacent cells, and ultimately determine the fate of the cell. However, in most cases, it is not independent role of mutual coordination on the development of the cell through a lot of \The Wnt pathway is an important way to regulate cell proliferation and differentiation, it is involved in almost all processes of embryonic development, which plays a very important role. The research has important significance for stem cells, tissue engineering and related diseases such as Alzheimer's disease, the treatment of tumors and prevention of Notch and its related signaling pathways. The crosstalk between the Wnt and Notch signaling pathway has previously been considered to occur mainly at two levels: a horizontal Wnt protein can be combined in the extracellular domain of Notch, the result is suppressed the binding of the Delta and Notch. Another level in the Wnt signaling pathway the Dishevelled protein can bind to the carboxyl terminus of the Notch intracellular domain blocking Notch non-activated conformation by stabilizing the Notch signaling pathway. Recently some new found between the Wnt and Notch pathways may also exist on the third level of crosstalk, Alzheimer factors involved, but its exact mechanism is not very clear. In this experiment, we transfected Notchl green fluorescent protein expression vector, raised the the expression of Notchl embryonic stem cells to study changes in the expression of Notch, Wnt signaling pathway molecules designed to further explore the interaction between the Notch signaling pathway, Wnt signaling pathway possible molecular mechanisms, so we update understanding of complex signaling networks in cell development, to further understanding of the pathogenesis of certain diseases, beneficial to better diagnose and treat these diseases. Experimental methods under sterile conditions cultured fibroblasts isolated from mouse embryonic tissues, with the H-DMEM culture medium at 37 ° C, 5% CO cultured under a condition of 2 . Fibroblasts cultured to the 2nd generation, treated with mitomycin. Embryonic stem cells (Embryonic stem cells ESCs) recovery and inoculated in processing good trophoblast cells with conditioned medium (including the 0.071gβ-mercapto-ethanol, 1 ml of non-essential amino acids, 0.011 g sodium pyruvate, 15% fetal calf serum ) cultured under the same conditions. Pending stem cell state is stable, cationic liposome transfection method the of Notch1 green fluorescent protein expression vector Notch1-PEGFP-C1 into mouse embryonic stem cells, cells were divided into 3 groups: Notch1-PEGFP-C1 plasmid transfection group, PEGFP- C1 empty plasmid transfected group and the normal control group. Expression of the green fluorescent protein observed by fluorescence microscopy 24h after transfection; collected 48h after transfection purified ESCs, RT-PCR detection of Notch1, HES-1, PS-1 and GSK-3β gene level expression, single-factor analysis of variance The differences among the groups. Experimental results. Effect of transfection detection - 24 hours after transfection transfection group and idling group under a fluorescence microscope visible green fluorescent protein expression after transfection 48h RT-PCR results show Notch1 mRNA expression both in three groups of cells, transfection group amplification dark bands than idling and control groups. PCR quantitative analysis software SPSS10.0 statistical analysis software for the analysis of the data obtained show that: the transfection Group Notch1 mRNA expression was significantly higher than the idling and the normal group (p <0.05 n = 5), while Notch signaling downstream effector molecules directly the HES-1mRNA expression changes with the Notch1 similar, fully illustrated transfected with Notch1-PEGFP-C1 ESC successfully raised Notch1 mRNA expression; 2.Notch1 raised after GSK-3β, PS-1 changes - 48h RT-transfected PCR results showed that GSK-3β, PS-1 expression in each group ESCs; the transfection group GSK-3β amplification compared idling and normal groups dark, statistical analysis of the results show that the the transfected group GSK-3βmRNA expression levels than The other two groups were significantly higher (p <0.05 n = 5); PS-1 amplification bands between the three groups of cells similar to the brightness, statistical analysis showed that the expression, there was no significant difference between the three groups (p> 0.05 n = 5). Conclusion 1. Notch signaling pathway in ESCs transfected Notch1 transduction enhancement. 2.Notch signaling pathways can be enhanced through the crosstalk inhibition of the Wnt signaling pathway. 3.PS1 in two paths crosstalk may play the role of the transfer signal, and its expression at the mRNA level does not change.