中文摘要：

目的：采用稳定表达Rab5a及其显性负性突变体Rab5a N133I的巨噬细胞系RAW264.7,研究Rab5a及其突变体过表达后对Cp G诱导的炎性细胞因子和Ⅰ型干扰素的表达的影响。方法：通过脂质体法将Rab5a及其突变体Rab5a N133I的真核表达载体转染到RAW264.7细胞中,用G418选择性培养基进行筛选,建立稳定表达Rab5a、Rab5a N133I的细胞系。通过RT-PCR、Real time-PCR和Western blot方法鉴定稳定表达的细胞系。用Cp G刺激稳定表达Rab5a、Rab5a N133I的细胞系8 h后检测细胞因子TNF-α、IL-1β和IFN-β的表达量变化。结果：RAW264.7转染Rab5a真核表达载体后Rab5a的表达量显著高于对照质粒转染细胞（P〈0.05）。Rab5a过表达后,在Cp G刺激作用下RAW264.7分泌的TNF-α、IL-1β显著升高（P〈0.01）,IFN-β的分泌也显著升高（P〈0.05）,而Rab5a N133I过表达后上述细胞因子的分泌均有所恢复。结论：Rab5a促进了Cp G诱导的巨噬细胞中炎性细胞因子和Ⅰ型干扰素的表达,Rab5a可能是TLR9信号通路的正向调控蛋白。

英文摘要：

Objective：Using the maerophage eel1 lines RAW264. 7 stably expressing Rab5a and its dominant negative mutant RabSaN 1331 as models to analyze the effect and the mechanism of Rab5 a, Rab5 aN1331 on CpG-induced production of pro-inflammatory cytokines and type I IFN. Methods： The eukaryotic expression vectors of RabSa and RabSaN133I were transfected into RAW264. 7 cells by liposome, and screened with G418. The G418-resistant colonies were obtained and amplified. The transformed cell lines were i dentified by RT-PCR,Real time-PCR and Western blot. The production of cytokines were measured after transformed cell lines of Rab5a and RabSaN133I was stimulation with CpG for 8 h. Results： Rab5a expression in transfected cells was significantly higher than the control cell group （P〈0. 05）. Overexpression of RabSa significantly promoted the production of TNF-a, IL-113 （P〈0. 01 ） and IFN-13 （P〈O. 05 ） in CpG stimulated RAW264. 7. The production of cytokines was restored in Rab5aN133I transfected cell line. Conclusion： Rab5a promotes CpG-induced pro-inflammatory cytokines and type I IFN in macrophages,it may be act as a positive regulator in TLR9 signaling pathway.