体外诱导M1型大鼠肺泡巨噬细胞对弓形虫增殖的影响

Effect of Inducing Rat M1 Alveolar Macrophage on Toxoplasma gondii Proliferation in Vitro

目的探究体外诱导M1型大鼠肺泡巨噬细胞对弓形虫感染的影响。方法体外用细菌脂多糖(LPS)+干扰素-γ(IFN-γ)诱导大鼠肺泡巨噬细胞极化为M1型,用实时荧光定量PCR方法检测巨噬细胞极化因子的变化,Western blot检测M1型巨噬细胞标志性蛋白诱导性一氧化氮合酶(iNOS)的变化,进一步使用扫描电子显微镜技术对诱导效果进行鉴定。M1型巨噬细胞与弓形虫共同培养,Diff染色观察其对弓形虫感染增殖的影响。结果与对照组相比,LPS+IFN-γ刺激后,M1型巨噬细胞中相关因子肿瘤坏死因子-α(TNF-α)、iNOS、CXCL10 mRNA、CXCL11 mRNA的相对表达量在6h升高(P<0.01),随后下降。Western blot结果显示,正常NR8383细胞不表达i NOS,LPS+IFN-γ刺激后细胞开始表达iNOS,在24h表达最为明显。扫描电子显微镜下观察到LPS+IFN-γ刺激细胞24h后,细胞表面突触增多、变长,细胞体积变大。正常NR8383细胞、M1型大鼠肺泡巨噬细胞分别与弓形虫进行共培养24h,Diff染色后,正常NR8383细胞内可以看到明显弓形虫增殖,而M1型大鼠肺泡巨噬细胞内几乎观察不到弓形虫。结论 M1型大鼠肺泡巨噬细可以明显抑制弓形虫的感染与增殖。

Objective To investigate the effect of inducing M1 macrophage in vitro on Toxoplasma gondii infection.Methods The rat alveolar macrophages were stimulated to differentiate into M1 macrophages by LPS and IFN-γ.Real-time fluorescence quantitative PCR was used to detect the changes of macrophage polarization factors.Western blot was used to detect the changes of macrophage marker protein iNOS in M1 macrophage cells.M1 macrophages were co-cultured with Toxoplasma gondii.Diff staining was used to observe its effect on the proliferation of Toxoplasma gondii infection.Results Compared with the control group,the relative expression levels of TNF-α,iNOS,CXCL10 and CXCL11 mRNA of M1-type macrophage were significantly increased at 6h after stimulation by LPS and IFN-γ,and then decreased.Western blot results showed that normal NR8383 did not express iNOS,and the cells began to express iNOS after being stimulated with LPS and IFN-γ.The expression was most obvious at 24h.Under the scanning electron microscope,cells stimulated by LPS and IFN-γfor 24 hours showed synapses increased on the cell surface and became longer and the cell volume became larger.Normal NR8383 cells and M1 macrophages were co-cultured with Toxoplasma gondii for 24h respectively.After Diff staining,the proliferation of Toxoplasma gondii was observed in Normal NR8383 cells,whereas toxoplasma could not be observed in M1 macrophages.Conclusion M1 macrophage can significantly inhibit the proliferation and infection of Toxoplasma gondii.