RUNX3基因对RAW264.7巨噬细胞极化的调控作用

Regulation of RUNX3 gene on RAW264.7 macrophage polarization

目的探讨RUNX3对巨噬细胞极化过程的调节作用和可能机制,为巨噬细胞参与的免疫炎症性疾病提供新的治疗思路。方法 (1)分别用IFN-γ、LPS和IL-4刺激RAW264.7细胞,使用RT-PCR方法检测刺激后细胞表面标志物arginase-1、iNOS的表达变化,观察刺激后RAW264.7细胞是否向M1或者M2方向极化;设定IFN-γ和LPS共刺激的细胞为M1组,IL-4刺激的细胞为M2组;另设正常对照组为M0组;(2)使用免疫荧光、RT-PCR方法检测各组细胞(M1、M2、M0)RUNX3表达情况;(3)建立RUNX3沉默的载体,沉默RAW264.7细胞系RUNX3基因,经G418选择性培养基筛选得到稳定表达的细胞系,RT-PCR检测细胞表面标志物iNOS和CD86变化,用ELISA法检测细胞分泌物TNF-α变化。结果 (1)用IFN-γ和LPS刺激RAW264.7细胞可使RAW264.7细胞向M1亚型极化,RT-PCR检测M1组细胞iNOS表达较M0组升高(P=0.002);而使用IL-4刺激RAW264.7细胞可使RAW264.7细胞向M2亚型极化;RT-PCR检测结果显示,M2组细胞arginase-1表达较M0组升高(P=0.021);(2)RUNX3mRNA在M1组细胞与M0组相比,表达增高(P=0.001),而在M2组细胞中表达降低(P=0.041);(3)沉默RUNX3后,CD86、iNOS mRNA表达较M0组降低(P=0.005),并且细胞分泌TNF-α减少(P<0.001)。结论 RUNX3转录活化可能促进巨噬细胞向M1型极化。

Objective To investigate the role of RUNX3 in the regulation of macrophage polarization so as to provide a new therapeutic approach for immunity-related diseases.Methods(1) After RAW264.7 cells were stimulated by IFN-γ,LPS and IL-4,respectively,the expressions of their surface markers(arginase-1 and iNOS)were detected by RT-PCR to observe whether RAW264.7 cells polarized to M1 or M2 after stimulation by IFN-γ,LPS and IL-4.The cells stimulated by IFN-γand LPS were named group M1 and those stimulated by IL-4 were group M2;the control group was group M0.(2) The expression of RUNX3 was detected by immunofluorescence and RT-PCR methods in each cell group(M1,M2 and M0).(3) RUNX3 over-expression vector was established.The RUNX3 gene in RAW264.7 cells was silenced.Cell lines with stable expression were screened with G418 culture medium.The expressions of cell surface markers iNOS and CD86 were detected by RT-PCR;cell secretion(TNF-α)was detected using ELESA method.Results(1) Stimulation of RAW264.7 cells with IFN-γand LPS could induce RAW264.7 cells to polarize into M1 type macrophages.The mRNA expression of iNOS in M1 group was higher than that in group M0 detected by RT-PCR(P=0.002),while using IL-4 to stimulate RAW264.7 cells could induce RAW264.7 cells to polarize into M2 macrophages.The results of RT-PCR detection showed that the expression of arginase-1 was higher in M2 group than in group M0(P=0.021).(2) The expression of RUNX3 mRNA in the M1 cells group was higher than that in the M0 cells group(P=0.001),but the expression in the M2 cells group was decreased(P=0.041).(3) After silencing of RUNX3,the expressions of RAW264.7 cell surface markers CD86 and iNOS(P=0.005)and the cells secretion of TNF-α(P<0.001)were decreased compared with those in the control group.Conclusion RUNX3 transcriptional activation may promote the differentiation of macrophages into M1 type.