The Role Of ERK1/2and P³⁸ MAPK Signaling Pathways In BMP9Induced Myocardiocytes Differntition From C3H10T1/2Stem Cells

PART ONE: AMPLIFICATION OF pAdEasyGFP、pAdEasyBMP9AND THE INFECTION OF C3H10T1/2STEMCELLSObjective: pAdEasyGFP and pAdEasyBMP9were Amplified andcollected with HEK293cells to get the high-titer adenovirus.C3H10T1/2stem cells were infected by pAdEasyGFP and pAdEasyBMP9toimprove the high expression of GFP and BMP9.Method:Amplify and collect HEK293cells to get the high-titerpAdEasyGFP and pAdEasyBMP9. infected by pAdEasyGFP and pAdEasyBMP9when C3H10T1/2stem cells passaged to about60%,cellmedium was changed after8hours,expression of the green fluorescence protein of the stem cells observed by inverted fluorescence microscope and detected infection efficiency by flow cytometry.Result:The adenovirus pAdEasyGFP and pAdEasyBMP9was amplified to high-titer, the pAdEasyBMP9infection efficiency of the C3H10T1/2stem cells have been detected as59.99%and42.16%at24 th hour and21st day.Conclusion:High-titer pAdEasyGFP and pAdEasyBMP9could be got with HEK293cells by amplifying repeatedly. High expressionof GFP and BMP9could be detected in C3H10T1/2stem cells after infected by pAdEasyGFP and pAdEasyBMP9. PART TWO:THE ACTIVATION OF ERK1/2AND P³⁸MAPKSIGNALING PATHWAY WHEN C3H10T1/2STEM CELLSTRANSFECTED BY pAdEasyBMP9Objective:To investigate the activation of ERK1/2and P³⁸MAPKsignaling pathway when C3H10T1/2stem cells transfected by pAdEasyBMP9.Method:The phospho-ERK1/2and ERK1/2,phospho-P³⁸MAPK and P³⁸MAPK were measured by Western blot,the phospho-ERK1/2andphospho-P³⁸MAPK was detected by Immunofluorescence at24th hour after the C3H10T1/2stem cells transfected by pAdEasyBMP9.Result:1.Transfected by pAdEasyBMP9produced a rapidly increase in phospho-ERK1/2and phospho-P³⁸MAPK and little change in ERK1/2and P³⁸MAPK.2.Phospho-ERK1/2and phospho-P³⁸MAPK irregular dispersed whole C3H10T1/2stem cells before transfected by pAdEasyBMP9or transfected by pAdEasyGFP;phospho-ERK1/2have different states thatmoved from cytoplasm to nucleus or only in nucleus，Phospho-P³⁸MAPK have different states that in nucleus and cytoplasm or only in nucleus after C3H10T1/2stem cells transfected by pAdEasyBMP9. Conclusion:ERK1/2and P³⁸MAPK signaling pathway can be activated when C3H10T1/2stem cells Transfected by pAdEasyBMP9. PART THREE:THE ROLE OF ERK1/2AND P³⁸MAPKSIGNALING PATHWAY IN BMP9INDUCEDMYOCARDIOCYTES DIFFERENTIATION FROM C3H10T1/2STEM CELLSObjective:To investigate the role of ERK1/2and P³⁸MAPK signaling pathway in BMP9induced myocardiocytes differentiation fromC3H10T1/2stem cells.Method:The cardiac-specific proteins cTnT and CX43were measured by Western blot, the cardiac-specific proteins cTnT,-MHC weremeasured by Immunofluorescence and the cardiac-specific gene GATA4and MEF2C expression were deteced by Real-time quantitative PCR at21st day after the C3H10T1/2stem cells transfected by pAdEasyBMP9and treated with U0126or SB203580.Result:U0126and SB203580significantly inhibited the pAdEasyBMP9-induced expression of CX43、cTnT、-MHC、GATA4、MEF2C in C3H10T1/2stem cells.Conclusion:ERK1/2and P³⁸MAPK signaling pathway can promote the BMP9induced myocardiocytes differentiation from C3H10T1/2stem cells.