稳定转染MiR-499对P19CL6细胞向心肌细胞分化的影响

小鼠miR-499基因包含在心肌重链肌球蛋白Myh7b基因的第19内含子中,并且在心肌细胞中特异表达,然而其在心肌细胞中表达的生物学功能和意义尚不清楚.利用可体外分化为心肌细胞的P19CL6细胞建立稳定表达miR-499的细胞株对研究miR-499的生物学功能具有重要意义.根据小鼠miR-499基因序列,设计PCR引物...     

hhlim促进DMSO诱导的P19细胞向心肌分化

为了确定hhlim是否参与胚胎期的心肌分化和发育过程,用可表达hhlim蛋白和hhlim反义RNA的真核表达质粒转染P19胚胎干细胞,经G418筛选得到稳定表达hhlim和hhlim反义RNA的P19细胞克隆后,观察hhlim对P19细胞向心肌分化和发育的影响.结果显示,Nkx2.5和GATA-4在未被外源性hhlim基因转染的P19细胞中不表达.DMSO刺激细胞2天后,GATA-4开始表达,3天后Nkx2.5的表达活性显著升高.hhlim的过表达不但有利于P19细胞的存活和生长,而且还可以使Nkx2.5和GATA-4的表达比对照细胞提前1天.反义hhlim细胞株被DMSO诱导5天后,细胞仍呈集落化生长.同时,Nkx2.5和GATA-4开始表达的时间明显延滞.结果表明,hhlim能促进P19细胞向心肌细胞分化,其作用是通过促进转录因子GATA-4和Nkx2.5的表达而实现的.     

Induction of a Hyaluronan Receptor,CD44, during Embryonal Carcinoma and Embryonic Stem Cell Differentiation

This paper describes the expression profile of the CD44 glycoprotein during differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. We have recently shown that CD44 is expressed in discrete embryonic structures and, in view of this, we sought an in vitro differentiation model of development in which we could study more readily the structure and function of the CD44 molecule. The P19 EC and CGR8 ES cells were chosen as they have the capacity to develop down the cardiac muscle pathway and we have previously demonstrated that CD44 is expressed abundantly in the embryonic myocardium. The differentiation process in both cell types is accompanied by an induction of CD44 mRNA and protein. However, in differentiated cultures CD44 is not expressed in contractile cells, indicating that these P19 cells do not represent CD44-positive embryonic cardiomyocytes. Expression of CD44 is observed on fibroblast-like cells which appear to migrate over and out from the plated aggregates. Hyaluronan, the major ligand for CD44, is also associated with these CD44-positive fibroblast-like cells. It is suggested that expression of both receptor and ligand by the fibroblastic cells is required for cell:matrix adhesion and cell motility. As CD44 is up-regulated in these cultures, P19 cells are now established as a useful model system to study the factors regulating expression of the CD44 gene.     

Neuronal and mesodermal differentiation of P19 embryonal carcinoma cells is characterized by expression of specific marker genes and modulated by activin and fibroblast growth factors

We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx-1 , goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose-dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA-responsive genes, including the three RA receptor genes (RARα, RARβ and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10⁻⁹mol/L RA or a factor produced by visceral endoderm-like cells (END-2 factor). The FGF effect is dose-independent. These results suggest an important function for RA and the END-2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development.     

Short-time glucose exposure of embryonic carcinoma cells impairs their function as terminally differentiated cardiomyocytes

The fetal and postnatal phenotype is influenced by developmental conditions experienced prenatally. Among prenatal development metabolic factors are of particular importance as they are supposed to predispose for pathophysiological alterations later in life and to pioneer functional impairment in senescence (metabolic programming). Till now the mechanisms of metabolic programming are not well understood. We have investigated various concentrations of glucose during differentiation of pluripotent P19 embryonic carcinoma cells (ECC) into cardiomyocytes. Undifferentiated P19 cells were exposed to 5mM (low), 25 mM (control), 40 mM or 100mM (high) glucose for 48 h during embryoid body (EB) formation, followed by plating and differentiation into cardiomyocytes in vitro with standard glucose supplementation (25 mM) for 10-15 days. The amount of cardiac clusters, the frequency of spontaneous beatings as well as the expression of metabolic and cardiac marker genes and their promoter methylation were measured. We observed a metabolic programming effect of glucose during cardiac differentiation. Whereas the number of beating clusters and the expression of the cardiac marker alpha myosin heavy chain (α-MHC) were comparable in all groups, the frequencies of beating clusters were significantly higher in the high glucose group compared to low glucose. However, neither the insulin receptor (IR) or insulin like growth factor 1 receptor (IGF1R) nor the metabolic gene glucose transporter 4 (GLUT4) were influenced in RNA expression or in promoter methylation. Our data indicate that a short time glucose stress during embryonic cell determination leads to lasting effects in terminally differentiated cell function.