谷氨酰胺对BCG诱导小鼠传代巨噬细胞凋亡的调控作用  

Regulation of BCG-induced Apoptosis in Murine Macrophages by Glutamine

作　　者：安琪 于嘉霖 吴晓玲 邓光存[1,2] AN Qi;YU Jialin;WU Xiaoling;DENG Guangcun(Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China,Ningxia University,Yinchuan 750021,China;School of Life Sciences,Ningxia University,Yinchuan 750021,China)

机构地区：[1]西部特色生物资源保护与利用教育部重点实验室(宁夏大学),银川750021 [2]宁夏大学生命科学学院,银川750021

出　　处：《畜牧兽医学报》2023年第7期3054-3063,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：宁夏自然科学基金(2022AAC02009);国家自然科学基金(32160162);宁夏科技创新领军人才培养项目(KJT2017002)。

摘　　要：旨在探讨谷氨酰胺在牛分枝杆菌卡介苗(Bacille Calmette-Guerin vaccine,BCG)感染巨噬细胞过程中对凋亡的调控作用。本文采用小鼠单核巨噬细胞RAW264.7,首先建立了BCG感染致细胞凋亡的细胞模型,采用Western blotting检测凋亡关键因子的表达变化,确定最佳感染条件;进而利用无谷氨酰胺培养基构建谷氨酰胺剥夺模型并结合BCG感染,采用ELISA、免疫印迹、免疫荧光和流式细胞术等技术检测了细胞内谷氨酰胺关键代谢指标的差异变化。通过上述研究,阐明谷氨酰胺对BCG诱导的巨噬细胞凋亡的调控作用,并初步探讨其机制。结果表明,BCG感染显著上调了小鼠巨噬细胞RAW264.7细胞中Caspase 3和PARP的蛋白表达(P<0.001),最佳感染时间为12 h,最佳感染复数为10 MOI,同时BCG感染促进了RAW264.7细胞内谷氨酰胺的分解代谢。剥夺谷氨酰胺后能极显著降低BCG感染后巨噬细胞中谷氨酸(P<0.001)和谷胱甘肽(P<0.01)的含量,极显著增加了细胞内ROS的含量(P<0.001)。同时,抑制谷氨酰胺代谢促进了RAW264.7细胞内促凋亡蛋白Caspase 3、PARP、Cytc和Bax的表达(P<0.001),极显著下调抑凋亡蛋白Bcl-2的表达(P<0.001)。综上表明,在BCG感染RAW264.7细胞的过程中,抑制谷氨酰胺代谢可以降低细胞内谷胱甘肽的含量,造成ROS的累积,进而通过内源性途径促进细胞凋亡。This experiment was to investigate the role of glutamine in the regulation of apoptosis in Bacille Calmette-Guerin vaccine(BCG)-infected macrophages.In this study,a cellular model of apoptosis caused by BCG infection was established using mouse mononuclear macrophages RAW264.7,and the optimal time of BCG infection and the number of infection replicates were clarified.Then,a glutamine deprivation model was constructed by glutamine-free medium and combined with BCG infection,and the Key metabolic markers of glutamine were detected by ELISA,immunoblotting,immunofluorescence and flow cytometry.The results showed that BCG infection significantly upregulated the expression of Caspase 3 and PARP in mouse macrophages RAW264.7(P<0.001)and promoted the catabolism of glutamine.Glutamine deprivation significantly reduced glutamate(P<0.001)and glutathione(P<0.01)levels in BCG-infected macrophages and increased intracellular ROS content(P<0.001).Meanwhile,glutamine deprivation highly significantly up-regulated the expression of Caspase 3,PARP,Cytc and Bax(P<0.001)and highly significantly down-regulated the expression of the apoptosis suppressor protein Bcl-2(P<0.001).These results indicated that glutamine deprivation in BCG-infected RAW264.7 cells promotes apoptosis by reducing intracellular glutathione levels,causing ROS accumulation and thus promoting apoptosis through an endogenous pathway.

关 键 词：BCG 谷氨酰胺 巨噬细胞RAW264.7 细胞凋亡 

分 类 号：S855.2[农业科学—临床兽医学]