流体剪切应力和雷洛昔芬对成骨细胞增殖的影响及作用机制研究

Fluid Shear Stress and Raloxifene Stimulates the Proliferation of Osteoblast Through Regulating the Expresstion of β-catenin and Estrogen Receptor α

目的研究流体剪切应力与选择性雌激素受体调节剂——雷洛昔芬单独作用及联合作用对体外培养的小鼠前成骨细胞MC3T3-E1细胞增殖的影响及其作用机制。方法选择12dyn/cm2(1dyn=10-5 N)流体剪切应力与10-7 mol/L雷洛昔芬单独作用和联合作用于体外培养的MC3T3-E1细胞,同时设置阴性对照组(雌激素拮抗剂)和空白对照组,倒置显微镜下观察细胞的外形、大小、细胞核的形态等,并进行活细胞计数,分别采用RTPCR和Western bolt方法检测细胞中β-catenin、雌激素受体α(ERα)的mRNA和蛋白的表达。结果相对于空白对照和阴性对照组,流体剪切应力组、雷洛昔芬组与雷洛昔芬+流体剪切应力组的细胞数量均增加,ERαmRNA和蛋白表达增加(P<0.05),而β-catenin mRNA和蛋白表达,除雷洛昔芬组的β-catenin mRNA表达差异无统计学意义(P>0.05)外,其余表达均增加(P<0.05)。雷洛昔芬组与流体剪切应力单独作用组的细胞数、β-catenin蛋白、ERαmRNA及蛋白表达水平之间差异无统计学意义(P>0.05),但雷洛昔芬+流体剪切应力联合作用组所有检测参数均高于前述两组(P<0.05)。结论流体剪切应力和雷洛昔芬均有促MC3T3-E1细胞增殖的作用,能上调β-catenin、ERα的表达,且两者联合作用后有协同效应。流体剪切应力和雷洛昔芬可能通过调节Wnt/β-catenin和ER信号通路来影响成骨细胞的增殖。

Objective To investigate how the fluid shear stress and raloxifene alone or in combination affect the proliferation of murine pre-osteoblast MC3T3-E1.Methods MC3T3-E1 cells cultured in vitro were treated with the fluid shearing stress and raloxifene alone or in combination,and the MC3T3-E1 cells without treatment were set as control（O group）,treated with estrogen receptor antagonist ICI-182780（10^-7 mol/L,I group）were set as negative control 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 ofβ-catenin and estrogen receptor alpha（ERα）were detected by reverse transcription polymerase chain reaction（RT-PCR）and Western blot respectively.Results ① As compared with those of the control group and negative control group,the cell proliferation,the expression of ERαmRNA and protein,and protein expression of β-catenin significantly increased in the groups of raloxifene,fluid shearing stress and raloxifene combination fluid shearing stress（P〈0.05）.RTPCR analysis showed that the levels ofβ-catenin mRNA though were not changed significantly in raloxifene group compared with O group（P〉0.05）,increased significantly in groups of fluid shear stress and raloxifene combination fluid shear stress.② The cells number,the expression of β-catenin and ERαwere not changed significantly between raloxifene group and fluid shearing stress group（P〉0.05）,but all test parameters in groups of raloxifene combination fluid shearing stress were higher than that of the previous two groups（P〈0.05）.Conclusion Fluid shear stress and raloxifene,the selective estrogen receptor modulator,were verifed to be able to promote MC3T3-E1 proliferation,increase the expression ofβ-catenin and ERα,and the combination functioned synergicly significantly.Fluid shear stress and raloxifene may possiblely affect the osteoblast proliferation by regulating the signaling pathways of Wnt/β-catenin and ER.