目的 探讨抗弓形虫药物乙胺嘧啶对巨噬细胞叶酸水平和免疫功能的影响。 方法 将小鼠巨噬细胞RAW264.7（1 × 10 ⁴个/孔）接种于96孔板中,用0（对照组）、0.01、0.05、0.10、0.50、1.00、5.00、10.00、50.00、100.00、500.00和1 000.00 μmol/L的乙胺嘧啶分别处理24 h后,CCK8检测细胞活性抑制率。将RAW264.7细胞（5 × 10 ⁵个/孔）接种于6孔板中,弓形虫RH株速殖子感染后3 h,用0（对照组）、0.10、1.00、10.00和100.00 μmol/L的乙胺嘧啶分别处理24 h,实时荧光定量PCR（qRT-PCR）检测弓形虫速殖子的二氢叶酸还原酶的表达。TRIzol试剂提取RAW264.7细胞的总RNA,qRT-PCR检测巨噬细胞二氢叶酸还原酶和炎症因子基因的表达,Western blotting检测巨噬细胞DHFR的蛋白表达,全自动化学发光免疫分析仪检测胞内活性叶酸水平。用BLAST对速殖子DHFR蛋白和小鼠巨噬细胞DHFR蛋白的氨基酸序列进行同源比对。用无叶酸培养基预处理RAW264.7细胞48 h,随后用100 μmol/L的乙胺嘧啶处理24 h,qRT-PCR检测无叶酸条件下巨噬细胞炎症因子基因的表达。两组之间比较采用t检验,多重比较采用单因素方差分析。 结果 CCK8检测结果显示,乙胺嘧啶的细胞活性抑制率具有剂量依赖效应,在0.01~0.10 μmol/L时可促进巨噬细胞的轻度增殖,浓度高于0.10 μmol/L时显示有抑制细胞增殖作用。qRT-PCR检测结果显示,0.10、1.00、10.00和100.00 μmol/L组弓形虫二氢叶酸还原酶mRNA相对转录水平分别为1.190 ± 0.054、1.460 ± 0.206、0.468 ± 0.077和0.399 ± 0.073,1 μmol/L组高于对照组,10.00和100.00 μmol/L组均低于对照组（P < 0.01）;而巨噬细胞二氢叶酸还原酶mRNA相对转录水平分别为0.789 ± 0.505、1.820 ± 0.119、2.120 ± 0.140和2.080 ± 0.189,其中1.00、10.00和100.00 μmol/L组均高于对照组（P < 0.05或0.01）。Western blotting分析结果显示,100.00 μmol/L组巨噬细胞DHFR蛋白表达显著高于对照组。BLAST同源比对显示,速殖子DHFR蛋白和小鼠巨噬细胞DHFR蛋白的同源性仅为37.0%。化学发光免疫分析结果显示,100.00 μmol/L组巨噬细胞的胞内活性叶酸水平为（10.600 ± 2.930）nmol/L,高于对照组（P < 0.01）。100 μmol/L乙胺嘧啶处理后,巨噬细胞炎症相关指标TNF-α、iNOS、Arg-1和IL-1β的mRNA相对转录水平分别为2.230 ± 0.100、11.800 ± 1.350、3.610 ± 0.256和7.810 ± 0.987,均高于对照组（P < 0.05或0.01）。乙胺嘧啶处理后,无叶酸培养组RAW264.7细胞TNF-α、iNOS、Arg-1和IL-1β的mRNA相对转录水平分别为2.460 ± 0.026、1.330 ± 0.091、0.974 ± 0.141和0.997 ± 0.018,iNOS、Arg-1和IL-1β低于含叶酸正常组（P < 0.05或0.01）,TNF-α高于含叶酸正常组（P < 0.01）。 结论 乙胺嘧啶可以通过促进小鼠巨噬细胞二氢叶酸还原酶的表达,升高胞内活性叶酸水平,从而诱导巨噬细胞炎症因子基因表达。

Objective To explore the effects of pyrimethamine, an anti-Toxoplasma gondii drug, on folate level and immune function of macrophages. Methods RAW264.7 cells were seeded in 96-well plates (1 × 10⁴ cells/well) and treated with 0 (control), 0.01, 0.05, 0.10, 0.50, 1.00, 5.00, 10.00, 50.00, 100.00, 500.00 and 1 000.00 μmol/L pyrimethamine for 24 h, respectively, followed by CCK8 assay. RAW264.7 cells were seeded in 6-well plates (5 × 10⁵ cells/well), and infected with T. gondii tachyzoites for 3 h, followed by treatement with 0.10, 1.00, 10.00 and 100.00 μmol/L pyrimethamine for 24 h, respectively. The expression of dihydrofolate reductase (DHFR) in T. gondii tachyzoites was detected by qRT-PCR. Total RNA of RAW264.7 cells was extracted by TRIzol reagent. The expression of DHFR and inflammatory genes in macrophages was detected by qRT-PCR, the protein abundance of DHFR was detected by Western blotting and intracellular folate levels were detected by Automated Chemiluminescence Systems. The amino acid sequences of DHFR from T. gondii tachyzoites and from RAW264.7 cells were compared by BLAST. RAW264.7 cells were cultured in folate-free medium for 48 h, and then treated with 100 μmol/L pyrimethamine for 24 h to detect the expression of inflammatory genes by qRT-PCR. The comparison between two groups was performed by t-test, and multiple comparison was performed by one-way ANOVA. Results CCK8 detection showed that the inhibition rate of pyrimethamine on cell activity was in a dose-dependent manner, which could induce slight proliferation of macrophages at 0.01-0.10 μmol/L, while inhibited cell proliferation at the concentration higher than 0.10 μmol/L. qRT-PCR showed that the mRNA expression of DHFR in T. gondii tachyzoites in the 0.10, 1.00, 10.00 and 100.00 μmol/L groups were 1.190 ± 0.054, 1.460 ± 0.206, 0.468 ± 0.077 and 0.399 ± 0.073 respectively, showing mRNA expression in the 1.00 μmol/L group being higher than that in the control group, and the level in the 10.00 and 100.00 μmol/L groups lower than that in the control group (P < 0.01). Meanwhile, the mRNA expression of DHFR in the RAW264.7 cells were 0.789 ± 0.505, 1.820 ± 0.119, 2.120 ± 0.140 and 2.080 ± 0.189 respectively, higher in the 1.00, 10.00 and 100.00 μmol/L groups than those in the control group (P < 0.05 or 0.01). Western blotting results showed that the protein abundance of DHFR in macrophages in the 100.00 μmol/L group was significantly higher than that in the control group. BLAST results showed that the homology between tachyzoite DHFR and mouse macrophage DHFR was only 37.0%. Using Automated Chemiluminescence Systems, we found that the intracellular folate level of macrophages in the 100.00 μmol/L group was (10.600 ± 2.930) nmol/L, which was much higher than that in the control group (P < 0.01). After treatment with 100 μmol/L pyrimethamine, the mRNA expression of TNF-α, iNOS, Arg-1 and IL-1β were 2.230 ± 0.100, 11.800 ± 1.350, 3.610 ± 0.256 and 7.810 ± 0.987 respectively, which were all higher than those in the control group (P < 0.05 or 0.01). After treatment with 100 μmol/L pyrimethamine, the mRNA levels of TNF-α, iNOS, Arg-1 and IL-1β in the folate-free culture group were 2.460 ± 0.026, 1.330 ± 0.091, 0.974 ± 0.141 and 0.997 ± 0.018 respectively. The mRNA levels of iNOS, Arg-1 and IL-1β in the folate-free group were much lower than those in the normal folate group (P < 0.05 or 0.01), while the opposite was found for TNF-α (P < 0.01). Conclusion Pyrimethamine could increase the intracellular folate level of macrophages by promoting the expression of DHFR in mice, which further induces the inflammatory gene expression of macrophages.