Fluid Shear Stress And Raloxifene Stimulates The Proliferation Of Osteoblast Through Regulating The Expresstion Ofβ-Catenin And Estrogen Receptor α

Objective:to investigate how the fluid shearing stress alone and combination with raloxifene affect the proliferation of murine pre-osteoblast MC3T3-E1.Methods:MC3T3-E1cells were divided into six groups:(1) the cells were harvested after cultured72h as blank control group;(2) the cells were subjected to60minutes of12dyne/cm2fluid shearing stress by parallel plate flow chamber as the fluid shearing stress group;(3) the cells were treated with10-7mol/L ICI-182780for48hours as ICI-182780group;(4) the cells were stimulated with10"7mol/L raloxifene for48h as raloxifene group;(5) the cells were treated with10-7mol/L ICI-182780for48h and then pressurized with fluid shearing stress12dyne/cm2for60minutes as the ICI-182780conbination fluid shearing stress group;(6)and the cells were stimulated with10-7mol/L raloxifene for48h and then pressurized with fluid shearing stress12dyne/cm2for60minutes as the raloxifene conbination fluid shearing stress group. The cells were observed underoptical inverted microscope from cell shape, size, and nuclear shape to quantity respectively before harvested. The levels of mRNA and protein of beta-catenin and ER alpha were detected by reverse transcription polymerase chain reaction (RT-PCR) and western bloting respectively. All of the experimental data were analyzed by SPSS16.0software.Results:(1) the results of cell count showed that the cells number was increased significantly in groups of raloxifene, fluid shearing stress and raloxifene combination fluid shearing stress (P<0.05), decreased significantly in groups of ICI-182780and ICI-182780combination fluid shearing stress (P<0.05). The cells number was decreased in groups of raloxifene and fluid shearing stress, compared with the raloxifene combination fluid shearing stress group (P<0.05).(2) RT-PCR analysis showed that the levels of beta-catenin mRNA though were not changed significantly in raloxifene group compared with blank control group (P>0.05), increased significantly in groups of raloxifene combination fluid shear stress, fluid shear stress and ICI-182780combination fluid shear stress, but decreased significantly in ICI-182780group compared with blank control group (P<0.05); the levels of ER alpha mRNA were increased significantly in groups of raloxifene combination fluid shear stress, raloxifene and fluid shear stress, decreased significantly in ICI-182780group compared with blank control group (P<0.05), but not changed significantly in ICI-182780combination fluid shear stress group compared with blank control group (P>0.05). compared with the raloxifene combination fluid shearing stress group, the levels of beta-catenin and ER alpha mRNA were decreased in groups of raloxifene and fluid shearing stress(P<0.05).(3)The proteins levels of beta-catenin and ER alpha were consistent with he mRNA levels of them detected by RT-PCR, except the expression of ERa protein in raloxifene group was increased (VS the control, P<0.05), which was different with the RT-PCR results.Conclusion:(1) Fluid shear stress and Raloxifene, the selective estrogen receptor modulator, were verifed to be able to promote MC3T3-E1proliferation, the combination functioned synergicly. ICI-182780, the antiestrogen regulator, can inhibit MC3T3-E1proliferation.(2) Fluid shear stress and raloxifene alone or in combination can increase the expression of β-catenin and ERa, and the combination functioned synergicly significantly, while ICI-182780can decrease the expression of P-catenin and ERa.(3) Fluid shear stress and raloxifene may affect the osteoblast proliferation by regulating the signaling pathways of WNT/beta-catenin and ER.