The Effect Of Alcohol On Biological Characteristics Of Mesenchymal Stem Cells C3H10T1/2and On The Differentiation Of These Cells Into Cardiomyocyte-like Cell

Objective:Teratogenic effects of alcohol on heart development is already clear,but the mechanism is unclear, heart development is a extremely complexprocess, regulated by multi-factors, to explore the possible mechanism ofalcohol teratogenic effects, we have observed the biological characteristicsof mesenchymal stem cells C3H10T1/2and their differentiation intocardiomyocyte-like cells after the treatment of these cell with alcohol invitro.Methods:MTT assay was used to determine the cytotoxicity of alcohol onC3H10T1/2cells and select appropriate intervention concentrations ofalcohol, the growth curve was drawn by detecting cell absorbance atdifferent culture times. After the treatment of C3H10T1/2cells with thedifferent concentrations of alcohol for48h, we used flow cytometry to detectcell cycle, and used Q-PCR and Western blot to detect Oct-4expression levels. BMP2(Bone morphogenetic protein2) was used as the inducer toinvestigate the effect on C3H10T1/2cells differentiation intocardiomyocyte-like cells after the treatment of the cell with alcohol for24h,the cells were divided into four groups: BMP2group, alcohol+BMP2group,C3H10T1/2group and positive control group. Q-PCR was used to detectcardiac-specific genes GATA4and MEF2C expression levels,cardiac-specific proteins including cTnT and Cx43, were detected usingWestern blot at1,2,3,4weeks after the treatment.Results:C3H10T1/2cells grows well in a37℃,5%CO2cell incubator. Weselected50mM,100mM,200mM as intervention concentrations of alcoholby MTT assay, compared with the control group,50mM alcohol does notaffect cell proliferation, however200mM alcohol caused a significantinhibition to cell growth by about20%(P<0.05). cell cycle among thesegroups showed no significant difference (P>0.05)， however,200mMalcohol group showed a downward trend in G2phase, and a upward trend inS phase, G2/G1was increased, suggesting that s-phase arrest inhibit cellproliferation in200mM alcohol group; Oct-4gene and protein expressionlevels also showed no significant difference (P>0.05).In the process of differentiation, there were no significant differencebetween the BMP2induced group and alcohol+BMP2induced group on theexpression levels of GATA4and MEF2C (P>0.05), but the expression levels was still significantly lower than the positive control group (P <0.05),C3H10T1/2cells sustained low expression levels.The results of cTnT andCx43were similar to cardiac-specific genes, while no related proteins weredetected in C3H10T1/2cells.Conclusion:Alcohol inhibit the proliferation of C3H10T1/2cells in adose-dependent effect; it has not yet have a significant impact on the cellcycle, and also no significant differences were detected on the Oct-4expression levels. There was no significant effect on the differentiation ofthese cells into cardiomyocyte-like cells, suggesting that teratogenic effectsof alcohol may not by affecting Oct4expression and further affect the celldifferentiation.