阿奇霉素对弓形虫感染孕妇免疫细胞影响的体外实验研究

Effect of azithromycin on immune cells of pregnant women with Toxoplasma gondii infection: an in vitro experiment

目的探究阿奇霉素(azithromycin AZM)在体外对弓形虫(Toxoplasma gondii TOX)感染孕妇外周血单核细胞(peripheral blood mononuclear cells PBMCs)的免疫调节作用。方法采用密度梯度(Ficoll)法分离制备PBMCs,将绿色荧光蛋白(greenfluorescentprotein GFP)PLK弓形虫接种在PBMCs细胞上,将细胞分为PBMCs组和PBMCs+TOX组。使用60μg/ml AZM作用后,将PBMCs+TOX组分为对照组和AZM组。应用倒置荧光显微镜观察PBMCs+TOX组在60μg/ml阿奇霉素在不同作用时间点下的弓形虫增殖变化;利用免疫印迹法(western blot)及ELISA检测各处理组PBMCs细胞上免疫相关因子白细胞介素-12(Interleukin-12,IL-12)、干扰素-γ(interferon-γ,IFN-γ)和肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)蛋白表达水平及浓度;利用流式细胞仪检测各处理组PBMCs细胞亚群CD4+ T细胞PD-1阳性率及CD4+/CD8+变化。结果与其他作用时间点比较,阿奇霉素作用48 h抑制弓形虫增殖能力最强;与PBMCs组比较,PBMCs+TOX组PBMCs细胞IL-12、IFN-γ蛋白表达水平及浓度均下调,而TNF-α水平上调(P<0.05);AZM组PBMCs细胞IL-12、IFN-γ蛋白表达水平及浓度均高于对照组,而TNF-α低于对照组(P均<0.05);AZM组CD4+/CD8+T淋巴细胞比例上升,而CD4+ T细胞PD-1阳性率表达下降。结论阿奇霉素在体外抑制弓形虫增殖,其机制与介导弓形虫感染PBMCs孕妇细胞免疫保护有关。

OBJECTIVE To explore the in vitro immunoregulatory effect of azithromycin(AZM) on peripheral blood mononuclear cells(PBMCs) in pregnant women with Toxoplasma gondii infection. METHODS PBMCs were isolated by density gradient(Ficoll) method and inoculated with green fluorescent protein(GFP)PLK toxoplasma, which were then divided into the PBMCs group and the PBMCs+TOX group.After using 60 μg/ml azithromycin, the PBMCs+TOX group was divided into the control group and the AZM group after the treatment with 60 μg/ml of AZM.The proliferation of T.gondii of the PBMCs+TOX group was observed by inverted fluorescence microscope at the time points of treatment with 60 μg/ml of AZM;the levels of interleukin-12(IL-12), interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) in PBMCs were detected by means of western blot and ELISA;the positive rate of CD4+ T cell PD-1 and CD4+/CD8+ ratio in PBMCs subsets were determined by using flow cytometry. RESULTS As compared with other time points, AZM achieved the most powerful effect on inhibiting the proliferation of T.gondii after the treatment for 48 h.As compared with the PBMCs group, the levels of both IL-12 and IFN-γ in PBMCs of the PBMCs+TOX group were down-regulated, while the TNF-α level was up-regulated(P<0.05).The levels of IL-12 and IFN-γ in PBMCs of the AZM group were significantly higher than those of the control group, however, the TNF-α level of the AZM group were significantly lower than that of the control group(all P<0.05).The percentage of CD4+/CD8+T lymphocyte of the AZM group was increased, while the positive rate of CD4+ T cell PD-1 was decreased. CONCLUSION AZM may inhibit the proliferation of T. gondii in vitro, its inhibition mechanism is associated with the induced PBMCs immune protection of the pregnant women with T.gondii infection.