The Impact Of P38MAPK Signaling Pathway In BMP-13-induced Cardiomyocyte-like Differentiation From C3H10T1/2Cells

PART ONE:THE ROLE OF P38MAPK SIGNALINGPATHWAY IN BMP-13INDUCEDMYOCARDIOCYTES-LIKE DIFFERNTITION FROMC3H10T1/2CELLSObjective: To study p38MAPK signal pathway in BMP-13inducedC3H10T1/2cells into cardiomyocyte-like cells.Methods: High titers Ad-BMP-13, Ad-GFP were amplified by293cells.C3H10T1/2cells transfected with Ad-BMP-13and Ad-GFP for24hours, the expressions of green fluorescence were observed under invertedfluorescence microscope, transfection efficiency were detected by flowcytometry. Experimental groups were as follows: Ad-BMP-13transfectiongroup, Ad-GFP transfection group and C3H10blank group. Ad-BMP-13transfected C3H10T1/2cells for24hours, The p-p38MAPK andt-p38MAPK were detected by Western blot. The positioning of p-p38MAPKwas detected by immunofluorescence technique; Results:1.To obtain high titer Ad-BMP-13, Ad-GFP. through293cells afteramplification2. Ad-BMP-13, Ad-GFP transfected C3H10T1/2cells for24hours byflow cytometry after transfection efficiency of41%and45.12%respectively.3. p-p38MAPK expression in BMP-13induced group weresignificantly higher than the the adenovirus GFP group and C3H10blankgroup (P <0.05). There was no significant difference about t-p38MAPKexpression among the groups.4p-p38MAPK expression in Ad-BMP-13induction group wasdetected by immunofluorescence in the cytoplasm and nucleus, but mainlyseen in the nucleus.Conclusion:1. High titers of Ad-BMP-13, Ad-GFP transfected C3H10T1/2cells toobtain the high expression of BMP-13.2. P38MAPK signal pathway can be activated by Ad-BMP-13inC3H10T1/2cell differentiation process PART TWO： AD-SI-P38AND SB203580BLOCKINGP38MAPK DURING THE IMPACT OF BMP-13INDUCEDTHE CARDIOMYTE-LIKE DIFFERENTIATIONOFC3H10T1/2CELLSObjective: To study the Ad-si-p38and SB203580blocking P38MAPKduring the impact of BMP-13induced the cardiomyocyte-like differentiationof C3H10T1/2cellsMethods:1. High titers AD-Si-P38, Ad-si-NC were amplified by293cells.Expression of red fluorescence was observed under inverted fluorescencemicroscope after transfected with Ad-si-p38and Ad-si-NC24h.Experimental groups were as follows:①Si-P38group: Ad-si-P38transfected C3H10T1/2cells24h group;②si-NC group: Ad-si-NCtransfected transfected C3H10T1/2cells24h group;③C3H10group: notby any processing of C3H10T1/2cells. Expression of t-p38MAPK wasdetected by Western blot.2. Ad-si-p38blocking p38MAPK on the BMP-13induceddifferentiation:SI-p38+Ad-BMP-13transfected group, si-NC+Ad-BMP-13transfected group, si-NC+Ad-GFP transfection group and the C3H10blankgroup. Expression of cTnT and Cx43were detected by Western blotanalysis;Expression of GATA-4and MEF-2C mRNA were detected byquantitative PCR. 3.SB203580blocking p38MAPK on the BMP-13induceddifferentiation: DMSO+Ad-BMP-13transfected group and SB203580(2,5and10umol/l)+Ad-BMP-13transfected group. Expression of GATA-4andMEF-2C mRNA were detected by quantitative PCR.Results:1. After AD-SI-P38, Ad-si-NC transfected C3H10T1/2cells24h,transfection efficiency was observed under inverted fluorescencemicroscope about50%, Expression of t-P38MAPK in Ad-si-P38groupcompared with AD-Si-NC group and C3H10were significantly lower byWestern blot (P <0.05).2. Expression of cTnT, Cx43proteins si-NC+BMP-13group weresignificantly higher than si-NC group and blank group(P <0.05); cTnT, Cx43protein expression of SI-p38+BMP-13group were significantly lower thansi-NC/BMP-13group (P <0.05). GATA-4and MEF-2C of si-NC+BMP-13group were significantly higher than the Ad-si-NC group and blank group (P<0.05); GATA-4, MEF-2C of SI-p38+BMP-13group were significantlylower than the level of si-NC+BMP-13group (P <0.05).3After the cells treated with different concentrations SB203580(2,5and10μmol/l), GATA-4, MEF-2C expression of SB203580pretreated withBMP-13induction group was significantly lower than BMP13+DMSOgroup (P <0.05); GATA-4, MEF-2C gradually reduced with SB203580concentration increased, Conclusion:1.High titers of AD-si-p38, Ad-si-NC transfected C3H10T1/2cells tolow expression of p38MAPK.2.Activation of the p38MAPK pathway to promote C3H10T1/2cellsinto cardiomyocyte-like cells.