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为了探讨小分子GTP酶蛋白Rac1和Rac2在人单核细胞中趋化迁移以及还原型辅酶II(NADPH)氧化酶活性中的作用,采用小分子干扰siRNA对人单核细胞中RAC1、RAC2分别进行特异性抑制,采用实时定量PCR技术、免疫印迹技术在RNA和蛋白质水平上确认抑制效果,使用甲酰三肽(formyl-met-leu-phe,fMLP)、人单核细胞趋化因子(monocyte chemoattractant protein-1,MCP-1)诱导单核细胞趋化;用血清调理的酵母多糖(serum opsonized zymosan,ZOP)、佛波酯(phosphomolybdic acid,PMA)激活单核细胞NADPH氧化酶活性,诱导活性氧(Reactive oxygenspecies,ROS)产生,以此对Rac1和Rac2作用进行研究.结果表明,小分子干扰siRNA能够在mRNA水平和蛋白质水平分别有效抑制目的基因表达;使用Chamber assay方法发现,仅Rac1参与了fMLP、MCP-1诱导的人单核细胞趋化.Rac激活实验确证,Rac1参与MCP-1诱导的趋化;细胞色素C还原法表明,Rac1和Rac2均参与PMA和ZOP诱导人单核细胞ROS生成.在人单核细胞中,RAC1和RAC2基因沉默模型的成功建立以及初步研究显示,Rac1和Rac2的不同作用结果将为深入研究它们在人单核细胞中的功能奠定了良好基础.
In order to investigate the role of small molecule GTPases Rac1 and Rac2 in chemotaxis and the activity of reduced coenzyme II (NADPH) oxidase in human monocytes, small interfering siRNAs were used to detect RAC1 and RAC2 in human monocytes Specific inhibition was confirmed by real-time quantitative PCR using Western blotting at the RNA and protein levels. The inhibitory effect was evaluated using formyl-met-leu-phe (fMLP), monocyte chemoattractant (MCP-1, MCP-1) induced monocyte chemotaxis. Serum-conditioned zymosan (ZOP) and phosphomolybdic acid (PMA) activated monocyte NADPH oxidase activity and induced reactive oxygen species (Rac1) and Rac2 (Rac1 and Rac2) were studied.The results showed that small interfering siRNA could effectively inhibit the expression of target gene at mRNA level and protein level respectively.Using Chamber assay, it was found that only Rac1 was involved in fMLP , MCP-1 induced human monocyte chemotacticity.Rac activation experiments confirmed that Rac1 is involved in the chemotaxis induced by MCP-1; cytochrome C reduction method showed that Rac1 and Rac2 are involved in PMA and ZOP induced human Nuclear Cell ROS Generation The successful establishment and preliminary studies of the RAC1 and RAC2 gene silencing models in human monocytes show that the different effects of Rac1 and Rac2 will provide a good basis for further studies of their function in human monocytes .