TNF-α刺激大鼠骨髓间充质干细胞的机制研究

Mechanism of TNF-α in Stimulation of Bone Marrow Mesenchymal Stem Cells in Rats

目的:研究肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)刺激大鼠骨髓间充质干细胞(marrow-derived mesenchymalstem cells,MSCs)的作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,用TNF-α刺激骨髓间充质干细胞(MSCs),通过酶联免疫吸附剂测定法(enzyme linked immunosorbent assay,ELISA)观察比较不同组别细胞的生长因子分泌和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞的培养成功。②无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的生长因子分泌显著性增加,而通过磷酸化IκB的表达量显著性增加提示NF-κB被激活(P<0.05);同时TNF-α刺激组与TNF-α+NF-κB抑制剂组比较,TNF-α+NF-κB抑制剂组的生长因子分泌显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制(P<0.05)。结论:NF-κB对TNF-α刺激下的骨髓间充质干细胞分泌生长因子有关键性作用。

Objective：To investigate the promotive effects of NF-κB on the marrow-derived mesenchymal stem cells.Methods：Mononuclear cells were isolated from rat bone marrow by density-gradient centrifugation and were cultured on fibronectin-coated plates,supplied with bovine pituitary extract.MSCs were identified by Morphology and Surface molecule markers of MSCs.After the mar-row-derived mesenchymal stem cells were stimulated by TNF-α,the production of the growth factors and the protein expression of NF-κB in different groups were compared by ELISA and WESTERN BOLT.Results：① The marrow-derived mesenchymal stem cells were successful cultured by observing the morphology of the marrow-derived mesenchymal stem cells and detecting the cell surface antigen by flow cytometry assays.② The production of the growth factors had significantly increased in TNF-α stimulation group compared with that of no TNF-α stimulation group.The expression of p-IκB increased significantly indicating the activation of NF-κB（P0.05）.The production of the growth factors had significantly decreased in stimulation of TNF-α plus the inhibitor of NF-κB group compared stimulation of TNF-α group,.And the protein expression of p-IκB significantly decressed indicate the inhibition of NF-κB（P0.05）.Conclusion：NF-κB had crucially protective effects on the marrow-derived mesenchymal stem cells stimulated by TNF-α.