重组IFN-α-IL-18对鸡淋巴细胞组胺的诱导生成和NF-κB p65的激活

摘要

Objective:To discuss the effects of recombinant IFN-α-IL-18 fusion protein on chicken lymphocyte histamine induced and the NF-κB p65 activation and nucleo-cytoplasmic transport.Methods: The healthy chickens blood was sterile adopted with anticoagulant,then separation of the chicken peripheral blood lymphocyte and divided into 10 groups:The yeast expression and purification protein IFN-α-IL-18,IL-18,IFN-α were added with 250 ng/ml,500 ng/ml and 1 000 ng/ml respectively while the control was only added RPMI1640 with 3 repetitions for each group.Then the histidine decarboxylase activity,histamine,IFN-γ,PI3K,MAPK and NF-κB p65 in cell nucleus were detected.Results: The recombinant IFN-α-IL-18 and IL-18 could significantly promote the activity of histidine decarboxylase (P<0.01),increase the contents of histamine (P<0.01),induce IFN-γ (P<0.01),improve the contents of PI3K (P<0.01) and the NF-κB p65 levels in nucleus (P<0.01),and the higher concentration of IFN-α had a similar effect to lymphocytes.The effects of IFN-α-IL-18,IL-18 and IFN-α on MAPK was acratia.Conclusion: IFN-α-IL-18 and IL-18 can stimulate chicken peripheral blood lymphocyte populations increased histamine contents significantly and promote the induction of IFN-γ.IFN-α-IL-18,IL-18 and IFN-α increase PI3K expression in lymphocyte associated with the NF-κB activation and NF-κB p65 nucleo-cytoplasmic transport.The study built foundation for the function of IFN-α-IL-18 and the exploration of the mechanism of controlling epidemic diseases.%目的:探讨鸡IFN-α-IL-18融合蛋白对鸡外周血淋巴细胞组胺诱导产生和NF-κB p65的激活与转运.方法:无菌采取健康鸡抗凝血,分离鸡外周血淋巴细胞,以RPMI1640完全培养液调整细胞密度为5×106细胞/ml,将细胞分成10组,分别加入酵母表达并纯化的IFN-α-IL-18,IL-18和IFN-α蛋白终浓度250 ng/ml,500 ng/ml和 1 000 ng/ml,对照组细胞仅加RPMIl640培养基,每组各设3个重复,37℃、5%CO2培养箱培养48 h,分别测定淋巴细胞中组氨酸脱羧酶活性,组胺含量,IFN-γ,PI3K,MAPK和细胞核中NF-κB p65的含量.结果:检测结果表明,重组IFN-α-IL-18和IL-18均能显著促进鸡外周血淋巴细胞中组氨酸脱羧酶活性(P<0.01),增加组胺含量(P<0.01),诱生IFN-γ(P<0.01),提高PI3K(P<0.01)和细胞核中NF-κB p65的含量(P<0.01),较高浓度的IFN-α对淋巴细胞有类似的作用;IFN-α-IL-18、IL-18和IFN-α对鸡淋巴细胞中的MAPK作用不明显,仅在较高浓度下IFN-α-IL-18和IL-18对细胞中MAPK产生影响(P<0.05).结论:IFN-α-IL-18及IL-18能够明显促进淋巴细胞中组胺的生成,并由此加强IFN-γ的诱生;IFN-α-IL-18、IL-18和IFN-α可能通过促进淋巴细胞中PI3K的表达激活NF-κB并促进NF-κB p65的核质转运,这为进一步开展IFN-α-IL-18融合蛋白的功能研究及其在疫病防控中的作用机制探索奠定了基础.