TNF-α刺激下大鼠骨髓间充质干细胞凋亡的机制研究

TNF-αInduce Apoptosis in Bone Mesenchymal Stem Cells via NF-κB

目的:研究肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)诱导大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymalstem cells,BMSCs)凋亡是通过激活核转录因子-κB(Nuclear translocation of the transcription factor nuclear factor-κB,NF-κB)。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(BMSCs)。用TNF-α刺激骨髓间充质干细胞(BMSCs),通过FITC-Annexin V/PI双染色法观察比较不同组别细胞的凋亡和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的凋亡细胞显著性增加,而通过核内NF-κB的表达量显著性增加提示其被激活(P<0.05);②同时TNF-α刺激组与TNF-α+NF-κB抑制剂组比较,TNF-α+NF-κB抑制剂组的凋亡细胞显著减少,而通过核内NF-κB的表达量显著减少提示其活性被抑制(P<0.05)。结论:NF-κB对TNF-α刺激下的骨髓间充质干细胞存活有关键性作用。

Objective： To investigate the promotive effect of NF-κB in the bone marrow-derived mesenchymal stem cells stimulated TNF-α inducing apoptosis. Methods： Mononuclear cells derived from rat bone marrow were isolated by density-gradient centrifugation and were cultured on fibronectin-coated plates, supplied with bovine pituitary extract. After TNF-α stimulated the marrow-derived mesenchymal stem tells, and the rates of cell apoptosis and the protein expression of NF-κB in different groups were compared by FACS analysis after staining with Annexin V and propidium iodide （PI） and WESTERN BOLT. Results： ① Comparing no stimulation of TNF-α group with under stimulation of TNF-α group, the rates of cell apoptosis had significantly increased. And the protein expression of NF-K B in nuclear significantly increased which indicated the activation of it （P〈0.05）. ②Meanwhile, comparing stimulation of TNF-α group with stimulation of TNF-α plus the inhibitor of NF-κB group, the rates of cell apoptosis had significantly dcressed, And the protein expression of NF-κB in nuclear significantly decreased which indicated the inhibition of it （P〈0.05）. Conclusion： NF-κB had crucially promotion effects on the marrow-derived mesenchymal stem cells stimulated by TNF-α.