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目的:探讨肿瘤坏死因子-α(Tumor Necrosis Factor alpha,TNF-α)对小鼠骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells,BM-MSCs)成骨分化的影响。方法:以在促成骨分化培养基(Osteogenic media,OS)中培养的小鼠BM-MSCs作为对照,用含有TNF-α的OS处理小鼠BM-MSCs。第7天进行碱性磷酸酶染色,或培养第21天进行茜素红染色和定量分析。第7天时用一份细胞提取得到的mRNA通过实时定量RT-PCR测定Runx2和Osterix的mRNA表达,用另一份细胞得到的细胞裂解液通过SDS-PAGE来测定Runx2和Osterix的蛋白质表达。结果:TNF-α抑制碱性磷酸酶活性;TNF-α抑制矿化骨节的形成;TNF-α抑制Runx2和Osterix的mRNA和蛋白质表达。结论:TNF-α对小鼠BM-MSCs成骨分化的抑制,可能部分是通过抑制成骨分化中两个关键的转录因子Runx2和Osterix的mRNA和蛋白质表达来实现的。
Objective: To investigate the effect of tumor necrosis factor alpha (TNF-α) on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: Mouse BM-MSCs cultured in Osteogenic media (OS) served as controls and treated with OS containing TNF-α. Alkaline phosphatase staining was performed on the 7th day, and alizarin red staining and quantitative analysis was performed on the 21st day of the culture. MRNA extracted from one cell at day 7 Runx2 and Osterix mRNA expression was measured by real-time quantitative RT-PCR and protein expression of Runx2 and Osterix was determined by SDS-PAGE with cell lysate from one cell. Results: TNF-α inhibited alkaline phosphatase activity; TNF-α inhibited the formation of mineralized joints; TNF-α inhibited Runx2 and Osterix mRNA and protein expression. CONCLUSION: The inhibitory effect of TNF-α on the osteogenic differentiation of mouse BM-MSCs may be partly through inhibiting the mRNA and protein expression of Runx2 and Osterix, two key transcription factors in osteogenic differentiation.