雌二醇通过雌激素受体α/糖原合酶激酶-3β/β联蛋白信号通路调节成骨细胞增殖

Estradiol stimulates proliferation of osteoblast cells via ERα/GSK-3β/β-catenin signaling pathway

目的研究观察雌二醇对小鼠成骨细胞前体细胞增殖影响并研究经典wnt通路在其增殖过程中的作用。方法本研究利用小鼠胚胎成骨细胞前体细胞Mc3T3-E1为细胞模型,不同浓度雌二醇处理Mc3T3-E1细胞,四甲基偶氮唑蓝(MTT)检测细胞增殖率,蛋白印迹法和免疫荧光技术检测经典Wnt信号通路信号分子表达。结果研究表明不同浓度雌二醇处理Mc3T3-E1细胞,MTT显示随着雌二醇浓度增加,Mc3T3-E1细胞增殖率增加,其中以10-7 mol/L浓度处理后细胞获得最佳增殖率。蛋白印迹法结果显示随着雌激素浓度增加,小鼠Mc3T3-E1细胞中p-GSK-3β,β联蛋白(catenin),细胞周期蛋白(Cyclin)D1的表达增加。经雌激素受体(ER)α沉默后,相对于空白对照组小鼠Mc3T3-E1细胞p-GSK-3β,β-catenin及Cyclin D1表达下降,而ERβ沉默后,同对照组相比成骨细胞中各分子表达则无明显变化。结论雌二醇可通过ERα/GSK-3β/β-catenin信号通路调节小鼠成骨细胞增殖。

Objective To determine viability and proliferation of the Mc3T3-E1 cells treated with different increasing concentrations and time of estradiol and to detect the relationship between the estrogen receptors and the canonical Wnt signaling pathway in these cells.Methods Mc3T3-E1 cells were treated with different increasing concentrations（DMSO,10-9,10-8,10-7,10-6 mol/L）and time（1,3,5,7,9days）of estradiol.Then we examined the proliferation of these cells assessed by MTT.Western blotting examined the expression of key proteins（β-catenin,GSK-3β,p-GSK-3β/Ser 9and Cyclin D1）.Confocal immunofluorescence detected the nuclear import ofβ-catenin.Plasmid vector（plentilox 3.7）of ERs RNAi and packaged recombinant lentivirus were constructed.The Mc3T3-E1 cells had been transfected the modified virous vectors for 24 hand then been treated with estradiol for 120 min.Then Western blotting examined the expression levels of ERα,ERβ,β-catenin,p-GSK-3βand Cyclin D1 in Mc3T3-E1 cells.Results Estradiol treatment increases viability and proliferation of mouse Mc3T3-E1 cells.Western blotting results indicated the increased expression levels ofβ-catenin,p-GSK-3βand Cyclin D1 in Mc3T3-E1 cells along with increasing concentration of estradiol treatment.Confocal immunofluorescence microscopy showed an increase of the nucleusβ-catenin level with estradiol treatment.Using ERαand ERβsiRNA to interfere the ERs mRNA expression,the results of Western blotting indicated the expression ofβ-catenin,p-GSK-3βand Cyclin D1 were lower in ERαsiRNA treatment group,while it was higher in ERβsiRNA group.Conclusion Estradiol mediated ERαinduces preosteoblastic cell proliferation through activation of Wnt/β-catenin signaling.