Study On The Relationship Between FHL1 Gene And The Differentiation Of P19 Cells To Cardiac Myocytes

Objective:To investigate the changes of FHL1(Four and a half LIM domains protein 1)gene mRNA and protein expression during the differentiation of P19 cells to cardiac myocytes,to explore the effects of FHL1 gene overexpression on the differentiation of P19 cells to cardiac myocytes,and to analyze the relationship between FHL1 gene and the the differentiation of P 19 cells to cardiac myocytes.Methods:(1)P19 cells were cultured with 1%DMSO in suspension for 4 days to form cell aggregation.Then the aggregates were cultured on gelatin-coated culture dishes without DMSO to 14 days.The beating of cells was observed.Western Blot of Cardiac TroponinⅠ(cTnI)were used to identify cell differentiation into cardiac cells.Total RNA and protein were extracted from P19 cells during the process of differentiation at various time points.The expression levels of FHL1 gene mRNA and protein were evaluated by RT-PCR and Western Blot,respectively.(2)The FHL1 gene eukaryotic expression vector was constructed.FHL1 overexpressing transformats was transfected into P19 cells.Then the P19 cells were induced into cardiac cells with 1%DMSO.Total RNA was extracted from P19 cells during the process of differentiation at various time points.RT-PCR or Western Blot was performed to identify the mRNA expression levels of GATA-4 and MEF-2C gene or the protern expression levels of cTnI.Results:(1)After induced by DMSO for 4 days in suspension,spontaneous and rhythmically beating cells were present at 8 day,which were cTnI-positive.When P19 cells were induced to differentiate into cardiac myocytes,both the expression level of FHL1 gene mRNA and protein were up-regulated gradually From Day 0 to Day 8 of differentiation,the FHL 1 gene mRNA and protein expression were obviously up-regulated, there were significant differences between the different days(p＜0.05). From Day 8 to Day 14,both the mRNA and protein expression of FHL1 gene were kept in higher level,and there was no significant differences between different days(p＞0.05).(2)The eukaryotic expression vector of FHL1 gene were successfully constructed,witch was identified by colony PCR and restriction enzyme analysis.There was no significant differences in the mRNA expression levels of GATA-4,MEF-2c gene and in the expression level of cTnI protern between pcDNA^TM3.1/FHL1 and pcDNA^TM3.1 transfected P19 cells(p＜0.05). Conclusion:Both the expression levels of FHL1 gene and protein were up-regulated in the process of the differentiation of P19 cells into cardiacmyocytes.However,overexpression of FHL1 gene has no effect on the differentiation of P19 cells into cardiac myocytes.