【摘要】 目的：探讨扶正抗癌方（fuzheng kangai formula, FZKA）对小鼠M2型巨噬细胞RAW264.7极化的影响及其作用机制。方法：采用白细胞介素-4（interleukin-4, IL-4）作用RAW264.7细胞48 h建立巨噬细胞M2型极化模型，利用流式细胞术、实时荧光定量PCR、蛋白质印迹法检测M2型标志物巨噬细胞甘露糖受体（CD）206、CD163和转化生长因子-β（transforming growth factor-β, TGF-β）以及M1型标志物一氧化氮合成酶（inductible nitric oxide synthase, iNOS）的表达，将扶正抗癌方作用于M2型巨噬细胞后，检测M1及M2型巨噬细胞标志物、转录因子CCAAT增强子结合蛋白β（CCAAT enhancer-binding proteinβ, C/EBP-β）和miR155的表达，利用瞬时转染法将miRNA模拟物（mimics）和抑制剂（inhibitors）转入细胞后检测C/EBP-β及巨噬细胞极性的变化。结果：在IL-4(20 ng·mL^-1)作用下，CD206、CD163、TGF-β的表达增加（P<0.01）,iNOS表达降低（P<0.01）,IL-4成功诱导巨噬细胞向M2型极化；加入扶正抗癌方后CD206、CD163和TGF-β的表达降低（P<0.01）,iNOS表达增加（P<0.01）,扶正抗癌方可逆转M2型巨噬细胞的极化，同时C/EBP-βmRNA和蛋白的表达降低（P<0.01）,miR155的表达增加（P<0.01）;将miRNA的mimics和inhibitors转染至M2型巨噬细胞后miR155的表达分别增加和降低（P<0.01）,miR155 mimics和inhibitors转染成功；在M2型巨噬细胞中转入miR155 inhibitors后，C/EBP-βmRNA的表达增加（P<0.01）,miR155可靶向抑制C/EBP-βmRNA的表达；M2型巨噬细胞中转入miR155 inhibitors后加入扶正抗癌方，CD206和CD163表达增加（P<0.01,P<0.05）,miR155 inhibitors抑制了扶正抗癌方对M2型巨噬细胞极化的逆转。结论：扶正抗癌方可通过上调miR155的表达，进而靶向抑制C/EBP-β基因的表达，并进一步通过下调M2型巨噬细胞标志物CD206、CD163和TGF-β,上调M1型巨噬细胞标志物iNOS的表达，逆转M2型巨噬细胞的极化。更多还原

【Abstract】 Objective: To discuss the effects and mechanism of Fuzheng Kangai Formula（FZKA） on the polarization of mouse M2-type macrophage RAW264.7 cells. Methods: IL-4 was used to stimulate RAW264.7 cells for 48 hours to establish theM2-type macrophage model. The expression of the M2-type markers CD206, CD163 and TGF-β, and the M1-type marker iNOS were detected by flow cytometry, Quantitative Real-time PCR and western blot assay. The expression of M1 and M2-type macrophage markers, C/EBP-β and miR155 were detected after FZKA was applied to M2-type macrophages. The transient transfection was used to transfect miRNA mimics and inhibitors into cells, and then detected the changes of C/EBP-β expression and macrophage polarity. Results: It showed that IL-4(20 ng·mL^-1) could induce macrophages polarized to M2, which resulting that the expression of CD206, CD163 and TGF-β were increased（P<0.01）, while the expression of iNOS was decreased（P<0.01）. Under the intervention of FZKA, the expression of CD206, CD163 and TGF-β were decreased（P<0.01）, while the expression of iNOS was increased（P<0.01）.The polarization of M2-type macrophages could be reversed by FZKA. Meanwhile, the mRNA and protein expression of C/EBP-β was decreased（P<0.01）, while the expression of miR155 was increased（P<0.01）.When transfecting the mimics and inhibitors of miRNA into M2-type macrophages, the expression of miR155 was increased or decreased, respectively（P<0.01）. The miR155 mimics and inhibitors were transfected into the cells successfully. The mRNA expression of C/EBP-β was increased when the miR155 inhibitors were transfected into the M2-type macrophages（P<0.01）. The miR155 could inhibit the mRNA expression of C/EBP-β specifically. When the FZKA was added to M2-type macrophages which were transfected into miR155 inhibitors already, the expression of CD206 and CD163 were increased（P<0.01, P<0.05）. The miR155 inhibitors could inhibit the reversal of M2-type macrophage polarization by FZKA. Conclusion: FZKA can reverse the polarization of M2-type macrophage by up-regulating the expression of miR155, which targets to inhibit the expression of C/EBP-β, and further down-regulating the expression of the M2-type markers CD206, CD163 and TGF-β, up-regulating the expression of the M1-type marker iNOS.更多还原