ROP16过表达对THP-1诱导分化巨噬细胞极化的影响

Effect of ROP16 Overexpression on THP-1 Induced Differentiation of Macrophages

目的探讨弓形虫棒状体蛋白(ROP16)对人外周血单核细胞(THP-1)诱导分化巨噬细胞极化的影响。方法构建ROP16过表达慢病毒载体转染THP-1细胞,筛选得到稳转细胞系,使用佛波脂诱导分化为巨噬细胞,RT-PCR和Western blot分别检测相关mRNA及蛋白的表达情况。结果构建ROP16过表达载体,并转染THP-1筛选得到稳定过表达细胞系,佛波脂诱导分化为THP-1源性巨噬细胞。Western blot结果显示,M1型巨噬细胞的标志性蛋白一氧化氮合酶(iNOS)表达量升高,M2型巨噬细胞的标志型蛋白精氨酸酶-1(Arg-1)表达量降低(P均<0.05)。RT-PCR的结果显示,ROP16过表达组iNOS、白细胞介素-1(IL-1)、白细胞介素-12(IL-12)、肿瘤坏死因子-α(TNF-α)mRNA表达均高于空载体组和阴性对照组(P均<0.05);Arg-1、白细胞介素-10(IL-10)、转化生长因子-β(TGF-β)mRNA表达均低于空载体组和阴性对照组(P均<0.05)。结论弓形虫ROP16在THP-1分化巨噬细胞内过表达后可诱导细胞向M1型巨噬细胞方向极化。

Objective To investigate the effect of Toxoplasma gondii protein ROP16 on the polarization of human monocyte THP-1 induced differentiation in vitro.Methods THP-1 cells were transfected with ROP16 overexpressing lentiviral vector,and stably transfected cell lines were selected.Macrophages were induced by phorbol lipids.RT-PCR was used to detect the expression of related mRNAs and Western blot was used to detect the related proteins.Results A ROP16 overexpression vector was constructed and transfected with THP-1 to screen for stable overexpressing cell lines.Phorbol lipid induced differentiation into THP-1 derived macrophages.Western blot results showed that the expression of the marker protein iNOS of M1 macrophages increased,the expression of the marker protein Arg-1 of M2 macrophages decreased(P all<0.05).The results of RT-PCR showed that the expression of iNOS,IL-1,IL-12,and TNF-α mRNA in M1 macrophage-related factors was higher than that in the empty vector group and negative control group(P all<0.05).Compared with the empty vector group and the negative control group,the expression of macrophage-related factors Arg-1,IL-10,and TGF-β RNA decreased(P all<0.05).Conclusion Toxoplasma gondii ROP16 gene can be induced to polarize to M1-type macrophages after overexpression in THP-1 human monocyte-induced differentiated macrophages.