The Effect Of Knockdown PHC1 Protein On The Differentiation Of Mouse Embryonic Stem Cells Into Cardiomyocytes

Objective: To establish a model of mESCs differentiated into cardiomyocytes and investigate the effect of knockdown PHC1 protein on the differentiation of mESCs into cardiomyocytes.Methods: To knockdown PHC1 protein in mESCs.The expression of PHC1,multipotency and trigerm differentiation genes were detected by qRT-PCR,and the expression of PHC1 protein was detected by Western-Blot.The expression of PHC1,nanog protein was analyzed by Immunofluorescence.the mESCs were induced to differentiate into cardiomyocytes by Hanging Drop methods and 1%DMSO.The expression of specific genes in myocardial cells was detected by qRT-PCR and Western-Blot at different stages.Results:1.The effect of knockdown PHC1 protein on the multipotency in mESCs.The PHC1 shRNA was successfully constructed and infected into mESCs.Western-Blot results showed that the expression of PHC1 protein was significantly down-regulated compared with the control group,while qRT-PCR results showed that PHC1 and multipotent genes were down-regulated,especially nanog,and mesoderm-related genes were up-regulated.The results of immunofluorescence showed that the nanog protein in mESCs with PHC1 protein knockdown was significantly down-regulated,and it's has the co-localization phenomenon.The morphology of mESCs cells knocked down PHC1 protein showed obvious differentiation,but there was no obvious differentiation in the control group.2.The effect of knockdown PHC1 to mESCs differentiated into cardiomyocytes.The expression of NKX2.5,?-MHC,cTnT,MEF2 C and GATA4 in shPHC1 group were up-regulated during the differentiation of cardiomyocytes,and the percentage of Beating EB and cTnT were higher than the control group(P < 0.05).Conclusion:1.Knockdown PHC1 protein promotes the differentiation of mESCs into the mesoderm.2.Knockdown PHC1 protein promotes the differentiation of mESCs into cardiomyocytes and improves the differentiation efficiency.