巨噬细胞中诱导型一氧化氮合酶来源的NO对共培养HL60 细胞凋亡的影响

张　申; 尹利华; 卫涛涛

doi:10.3971/j.issn.1000-8578.3237

巨噬细胞中诱导型一氧化氮合酶来源的NO对共培养HL60 细胞凋亡的影响

Effects of Inducible Nitric Oxide Synthase Derived Nitric Oxide on Apoptosis of HL60 Cells Co-cultured with RAW264. 7 Macrophages

摘要

摘要: 目的研究巨噬细胞中诱导型一氧化氮合酶（iN—OS）来源的一氧化氮（NO）对共培养HL60细胞凋亡的影响。方法以脂多糖（LPS）和γ素（INF-γ）诱导RAW264．7巨噬细胞iNOS基因的表达产生过量NO为实验模型，通过噻唑蓝（MTT）试验、蛋白质印迹分析、荧光分析、流式细胞术（FCM）、透射电镜和DNA琼脂糖凝胶电泳等分析技术，观察No对共培养的HL60细胞存活率、bcl-2和bax蛋白表达、Caspase-3活性和细胞凋亡的影响。结果 RAW264．7巨噬细胞中iNOS来源的No对共培养HL60细胞能造成氧化损伤，降低细胞的存活率；bcl-2表达明显下降，而bax表达增加；激活Caspase-3和促进DNA的降解。结论巨噬细胞中iNoS来源的No在诱导细胞凋亡中发挥重要的作用。

Abstract: Objective 　To study effects of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on apoptosis of HL60 cells co2cultured with RAW 264. 7 macrophages. Methods 　Upon stimulation with lipopolysaccharide (LPS) and interferon-γ( IFN-γ), inducible nitric oxide synthase gene was expressed in RAW 264. 7 macrophages, which caused the consequent generation of nitric oxide. Effects of nitric oxide on HL60 cells viability, expression of bcl-2 and bax protein, activity of Caspase-3 and cell apoptosis were evaluated with MTT assay, Western blot analysis, fluorescence analysis, flow cytomet ry (FCM), transmission elect ron microscopy ( TEM) and DNA agarose gelelect rophoresis. Results 　The results showed that iNOS-derived nitric oxide caused oxidative damage of HL60 cells co-cultured with RAW 264. 7 macrophages, and decreased cell viability, and evidently reduced expression of bcl-2 and increased expression of bax, and induced activity of caspase-3 and DNA f ragmentation. Conclusion 　The result s suggested important effect of iNOS-derived nitric oxide on apoptosis of cells in RAW 264. 7 macrophages.

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