【摘要】 目的 探讨连接蛋白43（connexin 43, Cx43）是否能够调节吡啡尼酮（pirfenidone,PFD）介导的小鼠RAW264.7巨噬细胞M2型极化。方法 将体外培养的对数生长期小鼠RAW264.7巨噬细胞分为对照组（Control组）、PFD组、PFD+Cx43特异性阻断剂Gap19组（PFD+Gap19组）、吡啡尼酮+阴性慢病毒对照组（PFD+NC组）、吡啡尼酮+Cx43过表达慢病毒组（PFD+OE组）。采用慢病毒转染技术，加入含有过表达慢病毒的培养基，使Cx43在巨噬细胞中过表达。采用Western blot技术检测小鼠RAW264.7巨噬细胞Cx43和M2型极化标记物CD206、Arg-1的蛋白表达水平，采用倒置荧光显微镜观察小鼠RAW264.7巨噬细胞I型精氨酸酶（arginase1,Arg-1）和CD206的蛋白表达和定位，采用实时荧光定量PCR（qRT-PCR）技术检测Cx43,Arg-1和CD206的RNA表达水平。结果 PFD组中的Cx43蛋白表达与Control组相比降低；PFD组中的M2型极化标志物CD206和Arg-1与Control组相比大幅升高；与PFD组相比，在PFD+Gap19组中Arg-1和CD206的表达均显著升高；与PFD组相比，在PFD+NC组中Arg-1和CD206的表达无明显改变。与PFD+NC组相比，PFD+OE组中Arg-1和CD206的表达均显著降低。结论 PFD可以通过下调Cx43促进小鼠RAW264.7巨噬细胞向M2型极化。通过Gap19和过表达慢病毒调节Cx43的表达，也可调节巨噬细胞M2型极化。更多还原

【Abstract】 This study was designed to investigate the effect of Cx43 on pirfenidone(PFD)-induced M2polarization of mouse RAW264.7 macrophages. Macrophages were cultured in vitro and were divided into control group, PFD group, PFD+Cx43 specific blocker Gap19 group(PFD+Gap19 group), PFD + negative lentivirus control group(PFD+NC group), and PFD+Cx43 overexpressionlentiviral group(PFD+OE group). Cx43 wasoverexpressed by lentivirus transfection technique.Western blot was used to detect the protein expressionlevels of Cx43 and M2-type polarization markers CD206and Arg-1 in mouse RAW264.7 macrophages, whileinverted fluorescence microscopy was used to observethe protein expression and localization of Arg-1 andCD206 in mouse RAW264.7 macrophages. Furthermore, the RNA expression levels of Cx43 and Arg-1 were detected by quantitative real-time PCR(q RT-PCR). Datashowed that as compared with the control group, Cx43 expression in PFD group was decreased, and the M2-typepolarization markers CD206 and Arg-1 were significantly increased. Compared with PFD group, the expressionlevels of Arg-1 and CD206 in PFD+Gap19 group were significantly increased, while the expression levels of Arg-1and CD206 in the PFD+NC group were not significantly changed. Compared with PFD+NC group, the expressions ofArg-1 and CD206 in PFD+OE group were significantly decreased. In summary, PFD can promote M2-typepolarization of mouse RAW264.7 macrophages by down-regulating Cx43. Regulation Cx43 expression by Gap19 andoverexpressed lentvirus can also regulate M2-type polarization of macrophages.更多还原