鸡柔嫩艾美尔球虫对免疫细胞体外增殖及产生的免疫相关分子的影响

Effects of chicken Eimeria tenella coccidian on proliferation of immune cells in vitro and the production of immune related molecules

【目的】检测鸡柔嫩艾美尔球虫体外刺激的巨噬细胞和淋巴细胞活力及分泌的细胞因子目的是了解球虫对免疫细胞增殖及产生的免疫相关分子的影响。【方法】无菌条件分离鸡外周血和脾淋巴细胞,淋巴细胞和HD11巨噬细胞分别与鸡柔嫩艾美尔球虫子孢子体外共培养,采用MTT、Griess、ELISA、RT-PCR法、流式细胞术等方法检测细胞增殖、免疫相关分子水平。【结果】子孢子组鸡外周血和脾淋巴细胞增殖明显高于对照组(P<0.05);子孢子组HD11巨噬细胞NO和iNOS的分泌量明显增加(P<0.05),外周血和脾淋巴细胞NO和iNOS分泌量升高,但统计差异不显著,脾淋巴细胞iNOS分泌量低于对照组差异不显著,但子孢子组免疫细胞上iNOS基因表达水平上调且显著高于对照组(P<0.05);子孢子组HD11巨噬细胞和外周血淋巴细胞IL-2分泌量极显著低于对照组(P<0.01),且IL-2mRNA表达下调;HD11巨噬细胞和脾淋巴细胞IFN-γ量极明显高于对照组(P<0.01),而外周血淋巴细胞IFN-γ分泌量明显降低,IFN-γmRNA表达量与对照组无显著统计差异;子孢子组HD11巨噬细胞吞噬能力及明显强于对照组(P<0.01),其表达MHC-Ⅱ分子的阳性细胞百分率显著高于对照组,细胞上表达KUL01表面分子阳性百分率显著降低,但阳性细胞平均荧光强度都极明显高于对照组(P<0.01)。【结论】鸡柔嫩艾美尔球虫刺激免疫细胞增殖,提高巨噬细胞和淋巴细胞NO和iNOS的分泌量明显增加及iNOS基因表达水平上调,但IL-2分泌量下降其mRNA表达下调,免疫相关分子(MHC-II)表达量增加,结果表明鸡柔嫩艾美尔球虫能不同程度影响免疫细胞增殖及产生免疫相关分子。

Objective The experiment was aimed to investigate the effects of chicken Eimeria tenella coccidian on the proliferation and secretion of immune cells and the expression of some surface molecules in them in vitro.Methods The peripheral blood and spleen lymphocytes were separated from chicken by sterile condition,then,these samples and HD11 macrophages were cultured with sporozoites of chicken coccidian in vitro respectively.MTT,Griess,ELISA and RT-PCR as well as flow cytometry were used to detect cell proliferation and secretion,surface molecular gene expression of immune cells.Results comparing with the control group,the proliferation of the chicken peripheral blood and splenic lymphocyte in sporozoite group were significantly increased.The NO level secreted from HD11 macrophages,peripheral blood,spleen lymphocyte,and that in iNOS level from HD11 macrophages were all significantly increased.The levels of iNOS in blood and splenic lymphocytes in sporozoite group were not significantly lower than those in the control one,and the expression of iNOS gene was significantly up-regulated.The IL-2 secretion and IL-2 mRNA expression levels in HD11 macrophages and peripheral blood lymphocytes in sporozoite group were much lower than those in control group.IFN-γlevels in HD11 macrophages and spleen lymphocytes increased significantly,but those mRNA levels in peripheral blood lymphocyte decreased.There was no significant differences of IFN-γgene expression between the sporozoite group and the control one.Phagocytes of HD11 macrophages and MHC-Ⅱpositive cell percentage in sporozoite group were significantly higher than those in the control group.There was a significant reduction in positive cell percentage of both expressing KUL01 and MHC-Ⅱsurface molecules,and KUL01 only expressing in the sporozoite group,but the average fluorescence intensity of positive cells were obviously higher than that in the control one.Conclusion It can be concluded that chicken coccidian could promote the proliferation and secretion of immune cells.