Rac1对巨噬细胞RAW264.7细胞生物学作用的影响

Effects of Rac1 on the biological behaviors of RAW264.7 cells

目的分析巨噬细胞RAW264.7的Rac1表达水平,通过构建Rac1载体和特异性siRNA探讨其对RAW264.7细胞生物学活性的影响。方法经反转录法克隆Rac1的cDNA并构建Rac1基因表达载体,Western blot技术检测Rac1的蛋白表达水平,定量荧光PCR(qRT-PCR)测定Rac1 mRNA,流式细胞术分析细胞表面膜分子表达的改变,Transwell法观察细胞的迁移能力,ELISA法检测细胞培养上清中相关细胞因子的表达。结果经测序等手段鉴定表明,Rac1载体构建成功,且将Rac1载体转入RAW264.7细胞可见其mRNA和蛋白表达均明显上调、细胞迁移能力增强,但对RAW264.7细胞膜表面CD80、CD86和MHC-Ⅱ类分子的表达没有明显影响。然而,Rac1抑制剂及其特异性siRNA却可上调上述3种膜分子的表达。此外,Rac1转染的RAW264.7细胞分泌的IL-1β水平显著低于siRNA处理组及Rac1抑制剂组,而IL-6、IL-33、TNF-α的表达各组之间没有显著差异。结论 Rac1的表达有助于RAW264.7细胞的迁移和抑制IL-1β的分泌,而对细胞的抗原提呈作用无显著影响。

This study was performed to analyze the expression level of Rac1 in RAW264.7 cells, and discuss the effect of Rac1 vector and specific si RNA on the biological activity of RAW264.7 cells. The reverse transcription method was used to clone Rac1 cDNA and construct Rac1 expression vector, and the protein expression level of Rac1 was detected by Western blotting. Reverse transcription-quantitative polymerase chain reaction（RT-q PCR）was used to determinate Rac1 mRNA, while flow cytometry analysis was used to detect the changes of CD80, CD86 and MHCⅡ expression, and cell migration ability was observed by transwell method. In addition, ELISA was used to detect the levels of cytokines in supernatants. Data showed that Rac1 vector was obtained, the expression of Rac1 mRNA/protein were significantly up-regulated in Rac1-transfected RAW264.7 cells, and the ability of migrationwas enhanced. But there were no significant changes in the expression of CD80, CD86 and MHC Ⅱ inRac1-transfected RAW264.7 cells, however, these three kinds of membrane molecules could be up-regulated byRac1 inhibitors or specific si RNA. Moreover, the IL-1β level in supernatants of Rac1-transfected RAW264.7 cellswas lower than that of other groups, but there were no difference in the levels of IL-6, IL-33 or TNF-α. Taken together, the positive regulation of Rac1 may contribute to the migration of RAW264.7 cells and inhibit the expression of IL-1β, but exert no significant effect on antigen presentation.