ToxoROP16Ⅰ/Ⅲ诱导M2型巨噬细胞偏移抑制M1分泌炎性细胞因子的研究

ToxoROP16Ⅰ/Ⅲ induces polarization to M2 phenotype of macrophages to inhibit M1 inflammatory cytokines study

目的研究慢病毒介导弓形虫效应分子ROP16 Ⅰ/Ⅲ( Toxo ROP16 Ⅰ/Ⅲ)经旁路途径诱导巨噬细胞RAW264.7(Mφ)向M2型巨噬细胞偏移(M2),抑制经典途径激活巨噬细胞(M1)炎性因子的产生。方法构建表达ROP16 Ⅰ/Ⅲ的重组慢病毒,转染Mφ,通过激活旁路途径向M2型细胞偏移。脂多糖(LPS)诱导Mφ通过经典途径向M1型巨噬细胞细胞偏移。M1和M2细胞进行混合培养。用qRT-PCR检测TNF-α、IL-1β、TGF-β1、IL-10、诱导型一氧化氮合酶(iNOS)和精氨酸酶1(Arg-1)的表达。Western blot检测ROP16 Ⅰ/Ⅲ、iNOS、Arg-1和PD-L2的蛋白表达。结果构建ROP16 Ⅰ/Ⅲ重组慢病毒并成功稳转Mφ,观察可见绿色荧光表达;Arg-1、PD-L2蛋白和TGF-β1、IL-10、Arg-1的mRNA表达升高,表明ROP16 Ⅰ/Ⅲ诱导了M2的偏移。LPS刺激后, qRT-PCR检测巨噬细胞的TNF-α和IL-1β mRNA表达上调,iNOS mRNA和蛋白表达同时升高,显示M1表型细胞特征。在M1和M2细胞混合培养中,M1型细胞分泌的上述促炎因子表达显著降低。结论 Toxo ROP16 Ⅰ/Ⅲ可驱动巨噬细胞向M2型偏移,能明显下调M1型细胞分泌炎性细胞因子。

Objective To investigate lentivirus mediated Toxoplasma gondii ROP16 Ⅰ/Ⅲ( Toxo ROP16 Ⅰ/Ⅲ) induced RAW264.7 macrophage polarization to M2-type macrophage by alternatively activated macrophages cells(M2) inhibits classically activated macrophages cells(M1) inflammatory cytokines. Methods Construction of recombinant lentivirus expressed ROP16 Ⅰ/Ⅲ, transfection of RAW264.7 by activated of the alternatively activated to M2 pheno- type macrophages. Lipopolysaccharide(LPS) induces RAW264.7 polarization to M1 phenotype macrophages by the classically activated pathway. M1 and M2 cells were mixed cultured. Expression of TNF-α, IL-1β, TGF-β1, IL-10, inducible nitric oxide(iNOS) and Arg-1 were detected by qRT-PCR. Expression of ROP16 Ⅰ/Ⅲ, iNOS, arginine 1(Arg-1) and PD-L2 were detected by Western blot. Results Recombinant lentivirus ROP16 Ⅰ/Ⅲ was stably transformed RAW264.7 and observed green fluorescent expression. The protein of Arg-1 and PD-L2 increased expression and TGF-β1, IL-10 and Arg-1 mRNA indicated that ROP16 Ⅰ/Ⅲ induced to M2 cells. TNF-α and IL-1β mRNA in macrophage were detected by qRT-PCR after stimulation with LPS, and iNOS mRNA and protein were up-regulated at the same time, which showed that induced to M1 phenotype macrophages. In the mixed culture of M1 and M2 cells, the above proinflammatory factors secreted by M1 cells decreased significantly. Conclusion Toxo ROP16 Ⅰ/Ⅲ can induce macrophages to M2 cells and down regulate the secretion of inflammatory cytokines by M1 cells.