The Change Of Hes-1,ps-1 And Gsk-3β's Expression In Mouse Escs After Transfection Of Notch1

BACKGROUNDNotch singnal pathway is a extraordinary conservative singnal transduction system in evolution. Notch singnal plays a key role in determing cells fate. Notch recepters enlarge and secure the differences between consecutive cells and eventually determine cells fate. However, most of the time it dose not work independently. The Notch singal pathway synergistically controls and regulates the growth, development and apoptosis of cells by lots of cosstalk with Wnt signal pathway etc. Wnt signal pathway is one of the most important pathway which regulate the proliferation and differentiation of cells. It participates and plays an extraordinary important role in almost all the process in embryo development. On the whole the research of Notch and related signal pathway is of greate sense to architecture project,whose focal point is stem cells and the treatement and prevention of related diseases such as Alzheimerse's disease and tumor.The crosstalk between Notch and Wnt signal pathway was once considered in two aspect: One is that Wnt protein may combine the ectodomain of Notch to inhibit the combination of Delta and Notch; the other is that Dishevelled protein in Wnt signal pathway may combine the carboxyl terminus in endodomain of Notch to firm the conformation of the non-activation of Notch , thus block up the Notch si'gnaf pathway. Latest discovery indicate that there may be a third aspect in the crosstalk between Notch and Wnt signal pathway, in which PS-1 plays an key role . Yet the concrete mechanisms is unclear.Therefore, in the study, we up-regulated the expression of Notch1 in ESCs by transfecting Notch1 green fluorescent protein(GFP) vector.Then detected the changes of related molecules in Notch and Wnt signal pathway, in order to approach the potential molecule mechanisms between the two signal pathway, and get new message of complicate signal network in cells during development. Thus know more about the pathogenesis of some disease, and preferably diagnosis and treat them afterward.METHODSEmbryonic fibroblast were isolated from the mouse embryonic tissue under aseptic conditions, then cultured with H-DMEM medium under the condition of 37℃, 5%CO₂. When embryonic fibroblast were cultured to the second generation, treated them with mitomycin. Embryonic stem cells were Reanimated and seeded on prepared fibroblast and cultured with conditioning medium (including 0.071gβ-mercaptoethanol, 1ml non-essential amino acid, 0.011g sodium pyruvate 15% fetal bovine serum ) under the same condition with embryonic fibroblast. When ESCs were in steady state, we use Lipofectamine 2000 transfected Notch1 green fluorescent protein(GFP) vector into them. The cells were divided into three groups:A .group of transfected with plasmid Notch1-PEGFP-C1; B .group of transfected with plasmid PEGFP-C1; C. group of normal ESCs. We observe the expression of green fluorescent protein in each group 24h after transfection under fluorescence microscope. Depurant ESCs were collected 48h after transfection. genetic expression of HES-1,PS—1 and GSK-3βin these cells were detected by RT-PCR. Statistic analysis: the experimental results were analyzed by SPSS 10.0 software with one-way variance analysis.RESULTS1. The expression of Notch1 and HES-1 were detected by RT-PCR. We find Notch1 mRNA PCR amplification strap in A group is brighter than group B and C. the expression of Notch1 in A group cells is obviously higher than control cells (p<0.05 n=5). The same change happened to HES-1, a downstream molecule of Notch1 signaling pathway. Our results demonstrate that we have successfullyup-regulate Notch1 in Notch signaling pathway in ESCs.2. We can observe that both GSK-3pand PS-1 are exist in ESCs ,we also find that thegene expression of GSK-3P down-regulated after tansfection with Notch1 (p<0.01n=5). However, the expression level of PS-1 by RT-PCR showed no differenceamong all the 3 groups . (p>0.05 n=5)CONCLUSION1. The transduction of Notch signal pathway is reinforced after transfection of Notch1.2. The reinforcement of Notch signal pathway can inhibit the Wnt signal pathway by crosstalk.3. PS-1 may exclusively transfer signal in the crosstalk between the two pathway. However, the expression of PS-1 in mRNA level dose not change.