Effects Of The P38MAPK Signaling Pathway On Bone Marrow Mesenchymal Stem Cells Differentiation Into Cardiomyocyte-like Cells Induced By Bone Morphogenetic Proteins-2

ObjectiveTo observe the effect of the differentiation of the bone marrow-derived stemcells(BMSCs) into cardiomyocyte-like cells with bone morphogeneticproteins-2(BMP-2),and further to investigate the effects of P38MAPK signalingpathway on the differentiation of the BMSCs induced with BMP-2towardcardiomyocyte-like cells.MethodsSD rat bone marrow-derived stem cells were isolated,cultured and amplifiedby whole bone marrow adherence method in vitro; The third passage cells weredivided into3groups:control group,BMP-2inducer group,BMP-2+P38MAPKblocker SB203580group,After24h induction,the medium was changed tocomplete culture medium without any inductor and the cells were culture for4weeks. The morphological observation was performed by the inverted phasecontrast microscope;the BMSCs were identified by immunofluorescencestaining;the changes of expression p-P38MAPK/t-P38MAPK in BMSCs indifferent times(15,30,60min) of induction were determined with Westernblotting;the immunohistochemical technique were used for detecting of theexpression of after4weeks of induced cell Cardiac specific troponin(cTnT)and connexin43(CX43).ResultsBMSCs morphological observation and identification：The majority of theprimary cells were round or oval shape, strong light refraction,drifted in culturemedium;The primary cells adherent growth after3days, Their differed in sizeand shape,and usually with processes which were different in diameter andlength.In7days,the cells were rapidly division and proliferation,the size andnumber of cells increased,and the irregular arrangement, spindle shape,colony-like growth of the cells.The third cell had larger size, morphologicalhomogeneous and irregular arrangement, their were spindle shape andexhibited a fibroblast-like. The identification of BMSCs:the expression of theSurface antigens CD90,CD44of BMSCs were positive,the respectively positiverate of the Surface antigens were97.64%and94.56%,Hematopoietic stem cellsurface antigen were negative. After4weeks induction, Cell morphology of thecontrol group did not change significantly, mostly were fibroblast-like andspindle shape. Blocker group morphological characteristics graduallychange,mostly short stick,and significantly wider,the size increases and the cellarrangement towards consistent,closely connected, visible muscle filament-likestructures, visible are widely distributed,some of the cells that end-to-endwas"bamboo-like the structure”,began to exhibit the characteristics of themuscle cells. Compared with the blocker group,the size of the inducer cellsincreases,cells extend pseudopodia connected, visible part muscle filament-like structures, but loosely arranged and inconsistent direction.The expression of p-P38MAPK and t-P38MAPK protein: the totalP38MAPK protein did not have significant difference in three groups; theexpression of p-P38MAPK protein: Inducer group, the protein level wasincreased at15min and was to reach a peak at30min and subsequentlydecreased at60min, the three points was a remarkable difference(P＜ 0.05));Compared with the control group,the protein level of the three points wassignificantly elevated in inducer group,the difference was significant(P＜0.05);Blocker group,the protein level was vary low at15,30and60min, there wasnot difference at the three points(P＞0.05);Compared with the inducer group,the protein level was significantly decreased in blocker group at the three points(P＜0.05),But compared with the control group,the protein level of the blockergroup was significantly elevated at three points(P＜0.05).The expression of Cardiac specific troponin(cTnT)and connexin43(CX43):After4weeks induction, the CX43of the control group was nagtive,but it had apositive expression in inducer and blocker group, the rate of differentiation ofthe Cardiomyocyte-like cells was11.32±1.9and18.73±4.1in two groups, thedifference was significan(tP＜0.05);The cTnT of the inducer and blocker groupwas positive expression(12.07±1.5vs19.17±2.2), displaying green fluorescence,Compared with the inducer group,the cTnT level was significantly elevated inblocker group(P＜0.05);the control group had a nagtive expression.ConclusionsBMP-2could induced diferentiation of BMSCs toward cardiomyocyte-likecells in vitro,and the P38MAPK sinaling pathway may plays a negativeregulating role during induction.