Rac1和Rac2参与调节人单核细胞趋化和NADPH氧化酶活性

Rac1 and Rac2 Are Involved in Chemotaxis and NADPH Oxidase Activity in Human Monocytes

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.

Rac1 and Rac2 are members of the small Rho GTPase family.They play essential roles in coordinating migration and superoxide production during phagocytes responses to chemo-attractants.Although earlier studies in Rac1 and Rac2 knockout mice have demonstrated the unique roles of each Rac isoform in chemotaxis and NADPH oxidase activation in neutrophils,it is unclear how human monocytes use Rac1 and Rac2 to achieve their immunological responses to foreign agent stimulation.Here,we used siRNA to suppress the expression of Rac1 and Rac2.Real-time PCR and Western-blot showed that Rac1 and Rac2 siRNA effectively suppressed their expression in mRNA and protein level,respectively.Chamber assay demonstrated that only Rac1 was involved in fMLP,MCP-1 triggered monocytes chemotaxis.Rac activation assay verified that MCP-1 induced Rac1 activation.Micro anion assay showed that both Rac1 and Rac2 were necessary for PMA and ZOP induced NADPH oxidase activity.Taken together,by suppression of Rac1 and Rac2,we provided evidence that Rac1 and Rac2 play different roles in chemo-taxis and NADPH oxidase activity in human monocytes.