雌激素受体α介导小鼠骨髓间充质干细胞成骨分化

Osteogenic differentiation of mouse bone marrowmesenchymal stem cells mediated by estrogen α receptor

目的：通过研究17β-雌二醇干预小鼠骨髓间充质干细胞（ MSCs）体外成骨分化，揭示雌激素预防和治疗绝经后骨质疏松症（PMO）的机制。方法贴壁法分离培养小鼠的MSCs，并通过流式细胞仪鉴定其细胞表面标志物，同时体外诱导分化，鉴定MSCs多向分化潜能；免疫荧光、RT-PCR和Western Blot检测MSCs表达雌激素受体α和β（ ER α和ER β）；在雌激素干预MSCs成骨诱导分化过程中，添加10-11～10-7 M的17β雌二醇和/或ER拮抗剂ICI182，780和ER α选择性拮抗剂MPP，采用冯·科萨染色和定量钙离子测定方法，检测矿化结节数量及钙离子浓度。结果体外培养的小鼠MSCs表达ER α和ER β。与对照组相比，17β雌二醇干预能显著增加矿化结节数量增加及钙离子浓度，ER拮抗剂ICI182，780和ERα选择拮抗剂MPP能逆转17β雌二醇的成骨诱导效应。结论17β雌二醇主要通过ER α能提高MSCs体外成骨诱导分化，可能是雌激素替代治疗PMO的实验依据。

Objective To illustrate the mechanism of estrogen in the prevention and treatment of postmenopausal osteoporosis （ PMO） through the intervention of 17β-estradiol in the osteogenic differentiation of mouse bone marrow mesenchymal stem cells （ MSCs） in vitro.Methods Mouse MSCs were cultured using adhesion method.The biomarkers on the cell membrane were identified using flow cytometry.After the induced differentiation in vitro, the multi-directional differentiation potential of MSCs was also identified.The expression of estrogen receptorαandβ（ ERαand ERβ） was detected using immunofluorescence, RT-PCR, and Western blotting.During the intervention of estrogen in the osteogenic differentiation of MSCs, 10-11-10-7M 17β-estradiol, ER antagonist, ICI182 and 780, and the specific antagonist of ER α, MPP, were additioned.The number of mineralized nodules and the concentration of calcium were detected using Von Kossa staining and the quantitative detection of calcium.Results The expression of ERαand ERβwas observed in mouse MSCs cultured in vitro.Compared with that in control group, the number of mineralized nodules and the concentration of calcium increased significantly after the intervention of 17β-estradiol, while the osteogenic induction effect of 17β-estradiol could be reversed by ER antagonist, ICI182 and 780, and the specific antagonist of ERα, MPP.Conclusion 17β-estradiol can improve the induction of osteogenic differentiation of MSCs in vitro mainly depending on ERα.This study may provide experimental basis for the estrogen replacement therapy in the treatment of PMO.