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w nt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W nt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 ,9 days) of estradiol .Then we ex‐amined the proliferation of these cells assessed by MTT .Western blotting examined the expression of key proteins (β‐catenin ,GSK‐3β,p‐GSK‐3β/Ser 9 and 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 h and 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 of β‐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 nucleus β‐catenin level with estradiol treatment .Using ERαand ERβsiRNA to interfere the ERs mRNA expression ,the results of Western blotting indicated the expression of β‐catenin ,p‐GSK‐3β and Cyclin D1 were lower in ERαsiRNA treatment group ,while it was higher in ERβsiRNA group .Conclusion Estra‐diol mediated ERαinduces preosteoblastic cell proliferation through activation of Wnt/β‐catenin signaling.
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