上海交通大学学报(医学版) ›› 2018, Vol. 38 ›› Issue (7): 745-.doi: 10.3969/j.issn.1674-8115.2018.07.005

• 论著·基础研究 • 上一篇    下一篇

雌激素对骨髓单核巨噬细胞增殖、凋亡和分化的影响

赵婧羽,黄明健,张晓玲   

  1. 上海交通大学医学院附属新华医院骨科,上海 200092
  • 出版日期:2018-07-28 发布日期:2018-07-30
  • 通讯作者: 张晓玲,电子信箱:xlzhang@shsmu.edu.cn。
  • 作者简介:赵婧羽(1992—),女,硕士生;电子信箱:zhaojingyu@sjtu.edu.cn。
  • 基金资助:
    国家自然科学基金(81772347)

Effects of estrogen on proliferation, apoptosis and differentiation of bone marrow macrophage

ZHAO Jing-yu, HUANG Ming-jian, ZHANG Xiao-ling   

  1. Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
  • Online:2018-07-28 Published:2018-07-30
  • Supported by:
    National Natural Science Foundation of China, 81772347

摘要: 目的·探究雌激素对骨髓单核巨噬细胞(bone marrow macrophage,BMM)的增殖、凋亡和破骨分化的影响及其作用机制。方法·分离正常C57BL/6J小鼠BMM,体外诱导分化为成熟的破骨细胞(osteoclast,OC)。外源性给予10-8 mol/L β-雌二醇的为实验组,同时加入雌二醇和1 μmol/L雌激素受体(estrogen receptor,ER)拮抗剂ICI-182780的为抑制剂组,另设对照组。用12周龄的C57BL/6J小鼠建立双侧卵巢摘除(ovariectomized,OVX)模型(OVX组,n5),同时设假手术组(sham组,n5)。术后3个月取其BMM体外培养并诱导破骨分化。利用Prestoblue检测BMM的增殖能力,TUNEL染色检测其凋亡情况,Western blotting检测凋亡相关蛋白caspase 3和caspase 8。实时定量PCR检测成熟OC标志物抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,Trap)和组织蛋白酶K(cathepsin K,Ctsk)mRNA水平的变化,细胞TRAP染色分析BMM破骨分化能力。Western blotting检测BMM被核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)激活的下游信号通路。结果·实验组BMM增殖能力比对照组弱,OVX组BMM增殖能力比sham组强。TUNEL染色显示实验组BMM中细胞凋亡率显著高于对照组,caspase 3和caspase 8的蛋白水平与该结果一致。PCR结果显示实验组Trap和Ctsk的mRNA水平显著低于对照组。OVX组的相关mRNA水平明显高于sham组。细胞TRAP染色和定量分析显示实验组比对照组的BMM破骨分化能力低,OVX组比sham组的破骨分化能力强。雌激素对BMM增殖、凋亡和破骨分化的影响均可被ER拮抗剂阻断。Western blotting结果显示,给予RANKL后实验组BMM的IκKα/β、p65、JNK磷酸化水平相比对照组均降低。结论·雌激素能抑制BMM增殖和破骨分化,促进BMM凋亡,且这一作用是通过ER介导的。

关键词: 骨髓单核巨噬细胞, 破骨细胞, 雌激素, 雌激素受体

Abstract: Objective · To investigate the effects of estrogen on proliferation, apoptosis and differentiation of bone marrow macrophage (BMM) and the mechanism. Methods · BMMs were isolated normal C57BL/6J mice and induced to differentiate to osteoclasts in vitro. BMMs in experimental group were administered with 10-8 mol/L exogenous estrogen and antagonist group with both estrogen and 1 μmol/L ICI-182780, an antagonist of estrogen receptor, and control group was designed as well. Five 12-week-old C57BL/6J mice underwent ovariectomy (OVX group) and sham group (n5) underwent sham surgery. All mice were sacrificed after 3 months to isolate BMM. Proliferation ability of BMM was assessed using Prestoblue, TUNEL assay was performed to detect apoptosis in each group. Caspase 3 and caspase 8 were detectedWestern blotting. Quantitative real time PCR was used to detect tartrate-resistant acid phosphatase (Trap) and cathepsin K (Ctsk) mRNA levels during osteoclastogenesis. TRAP staining of osteoclasts showed osteoclastogenesis ability. In addition, the downstream moleculars activatedreceptor activator for nuclear factor-κB ligand (RANKL) in BMM were detectedWestern blotting. Results · BMM multiplication ability was attenuated in experiment group compared with control group and it was stronger in OVX group than that in sham group. TUNEL assay showed that the apoptotic BMM in experimental group were more than those in control group and caspase 3 and caspase 8 were consisted with the results of TUNEL assay. PCR analysis showed that Trap and Ctsk mRNA levels significantly decreased in experiment group compared with control group. The mRNAs increased in OVX group in contrast to sham group. TRAP staining of osteoclasts and quantitative analysis showed that osteoclasts in experiment group were less than those in control group and osteoclasts in OVX group were more than those in sham group. The effects of estrogen on proliferation, apoptosis and differentiation of BMM were blockedantagonist of estrogen receptor. Western blotting showed that the phosphorylation of IκKα/β, p65and JNK activatedRANKL were attenuated in experimental group compared with that in control group. Conclusion · Estrogen inhibits proliferation and osteoclastogenesis of BMM, and aggravates their apoptosis through estrogen receptor.

Key words: bone marrow macrophage, osteoclast, estrogen, estrogen receptor

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