Fabp3 Gene With Myocardial Cell Differentiation

PartⅠExpressive changes of fat acid binding protein-3 gene during the differentiation of P19 cells to cardiac myocytesObjective:To investigate the changes of FABP3(Fat acid binding protein-3)gene mRNA and protein expression during the differentiation of P19 cells to cardiac myocytes,and to explore the relationship between FABP3 gene and the differentiation of cardiac myocytes.Methods:P19 cells were cultured with 1%DMSO in suspension for 4 days to form cell aggregation.Then the aggregates were plated on 6-cm cell culture dishes,and cultured without DMSO to 15 days.The beating of cells was observed and Western Blot of cardiac TroponinⅠ(cTnI)were used to identify cell differentiation.Total RNA and protein were extracted from P19 cells during the process of differentiation at various time points. The expression levels of FABP3 gene mRNA and protein were evaluated by RT-PCR and Western Blot,respectively.Results:After induced by DMSO for 4days in suspension,spontaneous and rhythmically beating cells were present within 10 days,which were cTnI-positive.When P19 cells were induced to differentiate to cardiac myocytes,the expression level of FABP3 gene mRNA was down-regulated gradually from day0 to day6 and there were significant differences between d0,d2-4 and d5-6(P＜0.05).But there was no significant differences for the expression level of FABP3 protein between d0 and d2-4,though the expression model of FABP3 protein during the differentiation of P19 cells was same as that of FABP3 gene mRNA with the lowest expression at d4 of the differentiation.The FABP3 gene mRNA expression was gradually up-regulated after d5,and there were significant differences between every two time points(d5-6,d8-10, d12-15,P＜0.05).Western Blot was coincident with the result of RT-PCR From d5 to d12 of the differentiation and there were significant differences between d5-6 and d8-12.Conclusion:The function of FABP3 gene might be involved in the differentiation of cardiac myocytes.PartⅡEffect of fat acid binding protein-3 gene overexpression on the differentiation of P19 cells to cardiac myocytesObjective:To investigate the effects of fat acid binding protein-3 gene overexpression on the differentiation of P19 cells to cardiac myocytes, and to explore the relationship between FABP3 gene and the differentiation of cardiac myocytes.Methods:The vector that expresses the fusion protein of fat acid binding protein-3 and green fluorescence protein(GFP)or FABP3 overexpressing transformats(FABP3-pcDNA3.1-myc/his-B)was constructed respectively. The pEGFP-N2 recombinant plasmid was transfected into P19 cells and the location of FABP3 expression in the transfected cells was observed by fluorescence microscope and confocal microscope.FABP3 overexpressing transformats was transfected into P19 cells.The P19 cells that overexpressed FABP3 were induced with 1%DMSO.The proliferation and apoptosis of P19 cell was analyzed by MTT method and flow cytometry.Total RNA were extracted from P19 cells during the process of differentiation at various time points.The expression levels of cTnT,alpha-MHC,GATA4,MEF2c gene mRNA were evaluated by RT-PCR.Results:Fluorescence microscope and cofocal imagery analysis with green fluorescent protein-tagged protein transiently expressed in P19 cells show that FABP3 localizes all over the cytoplasm and in the nuclei as well.But in the some time of splitting of cell,green fluorescence cannot find in the nuclei.MTT and cell cycle analysis of stably transfected cells found that FABP3 could inhibit proliferation of P19 cell and promote apoptosis of P19 cells.When analyzed the expression of cardiac muscle -specific molecular markers by RT-PCR assay in these cells during the differentiation,cTnT,alpha-MHC,GATA4,MEF2c whose expression are crucial during the development of heart were selected.Cell lines overexpressed FABP3 showed the lower expression levels of these marker genes when compared with the control cells at day 7,day8,day 10 and day 12.Conclusion:These results suggest that FABP3 may inhibit proliferation of P19 cell and promote apoptosis of P19 cells.The function of FABP3 gene might be involved in the differentiation of cardiac myocytes.