Effects Of Ethanol On The Apoptosis Of Osteoblasts

Objective:To examine the effects of ethanol on the apotosis of osteoblasts in vitro.Ethanol is capable of inducing apoptosis of osteoblasts.And the effect was demonstrated from many ways,including proliferation,differentiation and apoptosis.This achievement provide the theoretical basis for seeking for the approach of the prevention and treatment of bone damage caused by ethanol.Methods:1.Osteoblasts were obtained from newborn rat,and were cultured in vitro.Osteoblasts were distributed to two groups respectively:control group and treatment group.Cells were cultured with RPMI-1640 supplemented with 10%FBS.And the third generation of osteoblasts were used in study.Cells were cultured in 96-,12-and 6-well plates.The treatment group was treated with 100mM ethanol after 1d.2.The cell morphology were observed at 1d,3d,5d and 7d.MTT assay was used to test the cell proliferation.3.The indicators of osteoblasts differentiation,the ALP and BGP,were tested with ELISA at 3d,7d,10d and 14d.4.RT-PCR was used to analyed gene Bcl-2 and Bax which were related to apoptosis.5.Flow cytometry was used to check apoptosis at 1d after treatment by ethanol.Results:1.Osteoblasts proliferation:The osteoblast prolif-eration of control group strengthen gradually,there is significant difference between 5,7 d and 1,3d(P<0.05).But the ethanol group abate gradually,there is significant difference between 5,7 d and 1,3 d(P<0.05).Moreover,the cells proliferation of ethanol group were lower than control group at the corresponding period(P<0.05).2.Osteoblasts differentiation:The concentration of ALP and BGP increased in control group but decreased in ethanol group along with time(P<0.05),and the latter was lower than the former respectively at the corresponding period(P<0.05).3.Osteoblasts apoptosis detected on the level of gene:Compared to control group,The mRNA expression of Bcl-2 decreased,while Bax increased gradually when osteoblasts were treated 3d,7d,1Od and 14d by 10OmM ethanol.4.Osteoblasts apoptosis tested by flow cytometry:The apoptosis rate of treatment group[(2.94×0.91)%]detected by the flow cytometry at 1d after treatment by ethanol was higher than that of control group[(1.89×0.63)%,P<0.05].Conclusion:1.The osteoblasts proliferation and differentiation decreased obviously after treatment by ethanol.2.Ethanol is capable of inducing apoptosis of osteoblasts.