绝经后骨质疏松环境下TNF-α对骨髓间充质干细胞成骨分化的影响

Effect of TNF-α on osteogenic potential of BMSMCs in estrogen deficiency-induced osteoporosis

目的:探讨肿瘤坏死因子α(TNF-α)在绝经后骨质疏松的环境中对骨髓间充质干细胞(BMMSCs)成骨分化的影响。方法:建立绝经后骨质疏松小鼠模型(OVX组,卵巢摘除),通过Micro-CT、实时定量RT-PCR、茜素红和碱性磷酸酶(ALP)染色等技术比较假手术组小鼠(Sham组)和OVX小鼠BMMSCs的成骨能力,Elisa方法检测OVX小鼠体内TNF-α的表达水平。在成骨诱导的过程中,用10 ng/m L TNF-α刺激正常C57小鼠来源的BMMSCs,检测Runt相关转录因子2(Runx2)和Osterix等成骨基因的表达水平。去势后的C57小鼠通过尾静脉注射TNF-α中和抗体及空白对照,Micro-CT检测小鼠的骨量及情况。结果:成功建立小鼠OVX模型,OVX组骨密度明显低于Sham组,OVX组BMMSCs的成骨能力明显低于Sham组,OVX小鼠体内TNF-α水平升高;在体外TNF-α能够明显抑制BMMSCs成骨分化能力;注射TNF-α中和抗体后,能够下调小鼠体内TNF-α表达水平,增强小鼠股骨密度。结论:在小鼠雌激素缺乏所导致的骨质疏松环境下,TNF-α能够抑制BMMSC成骨分化能力,体内注射TNF-α中和抗体能够促进OVX小鼠骨形成,TNF-α抑制骨髓间充质干细胞成骨分化功能可能是绝经后骨质疏松形成的机理之一。

Objective：To investigate the effect of TNF-α in the osteoblastic differentiation of bone marrow mesenchymal stem cells （BMMSCs） derived from postmenopausal osteoporosis. Methods：Established an ovariectomized mouse model. Micro-CT was used to examine the bone in the femurs of the mice. The osteogenesis of BMMSCs derived from sham and OVX mice were investigated by ALP activity, ALP and Alizarin Red staining. Osteogenic genes Runx2 and Osterix were determined by real time RT-PCR and Western blot. TNF-α levels in serum were examined via Elisa assay. Results：The BMD of OVX group was significantly lower than that of Sham group. The osteogenic differentiation of OVX-BMSMCs was inhibited compared with Sham-BMMSCs. TNF-α levels were increased in OVX mice. TNF-α impaired BMMSCs osteogenic differentiation. TNF-α neutralizing antibody was injected via caudal vein in OVX mice and TNF-α levels were reduced. Calcein double labeling and Micro-CT showed bone formation were higher in OVX mice which were treated with TNF-α neutralizing antibody. Conclusions： TNF-α was a key inflammatory cytokine that inhibited osteogenic potential of BMMSC derived from estrogen deficiency-induced osteoporosis. Injection of TNF-α neutralizing antibody in vivo can increase bone formation of OVX mice. TNF-α impaired osteogenic differentiation may be one of mechanisms in estrogen deficiency-induced osteoporosis.