鸟分枝杆菌PPE25蛋白对THP-1巨噬细胞免疫效应的影响  

Effect of PPE25 protein of Mycobacterium avium on immune response of THP-1 macrophages

作　　者：宁雪萍 田爽 龙倩 何会姣 陈蕴玟 凌敏[1] NING Xue-ping;TIAN Shuang;LONG Qian;HE Hui-jiao;CHEN Yun-wen;LING Min(Key Laboratory of Longevity and Aging-related Diseases,Ministry of Education,China,Laboratory of Biological Molecular Medicine Research of Guangxi University,Department of Biotechnology,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学,长寿与老年相关疾病教育部重点实验室,广西高校生物分子医学研究重点实验室,广西医科大学生物技术教研室,广西南宁530021

出　　处：《生物技术》2021年第5期425-430,477,共7页Biotechnology

基　　金：广西自然科学基金项目(“鸟分枝杆菌PPE25调控巨噬细胞免疫应答的机制研究”,2018GXNSFAA281023)。

摘　　要：[目的]探讨鸟分枝杆菌PPE25在调控人源THP-1巨噬细胞免疫应答反应中的作用及机制。[方法]通过基因扩增、载体构建、诱导表达、分离纯化等过程制备鸟分枝杆菌重组PPE25蛋白;将重组PPE25蛋白作用于THP-1巨噬细胞后,利用流式细胞术检测巨噬细胞凋亡率;运用ELISA方法检测细胞上清中TNF-α的浓度;采取Western Blotting方法检测Bax、Bcl-2、TLR2、TLR4的蛋白表达水平;采用q PCR法检测IL-6、IL-12的mRNA表达水平。[结果]利用大肠杆菌表达系统成功制备重组PPE25蛋白;重组PPE25蛋白作用巨噬细胞后,细胞凋亡率升高(P <0.05),细胞中Bax蛋白表达水平升高(P <0.05),而Bcl-2蛋白表达水平降低(P <0.05);另外,细胞中TLR2、TLR4蛋白表达水平上升(P <0.05),TNF-α含量升高(P <0.05),IL-6及IL-12表达水平升高(P <0.05)。[结论]鸟分枝杆菌PPE25蛋白可以促进人源THP-1巨噬细胞凋亡,并可以通过激活巨噬细胞TLR2或TLR4信号途径促进细胞炎症因子IL-6及IL-12的分泌,从而诱导机体产生免疫应答,抵御鸟分枝杆菌感染。[Objective]To investigate the effect of recombinant protein PPE25 of Mycobacterium avium on immune response of human THP-1 macrophages and the molecular mechanism related. [Method]Gene amplification,vector construction,induced expression and separation and purification were used to prepare recombinant PPE25 protein of Mycobacterium avium. After treated the THP-1 macrophages with the recombinant PPE25 protein,flow cytometry was used to detect the apoptosis rate of macrophage. ELISA was utilized to detect the level of TNF-α in culture supernatant. Western Blotting was conduceted to measure the protein expression of Bax,Bcl-2,TLR2 and TLR4. The mRNA expression of IL-6,IL-12 were conduceted by q PCR.[Result]Recombinant PPE25 protein was successfully prepared by E. coli expression system. After treated with the recombinant PPE25 protein,the apoptosis rate of macrophages was increased( P < 0. 05),the expression of Bax proteins was increased( P <0. 05),and the expression of Bcl-2 protein was decreased( P < 0. 05). In addition,the expressions of TLR2 and TLR4 were increased( P < 0. 05),the level of TNF-α was increased( P < 0. 05),and the expression of IL-6 and IL-12 were increased( P < 0. 05).[Conclusion]The Mycobacterium avium PPE25 protein could enhance the apoptosis of human THP-1 macrophages,and promote the secretion of inflammatory cytokines IL-6 and IL-12 by activating the TLR2 or TLR4 signaling pathway in macrophages,so as to induce the body to produce immune response and resist Mycobacterium avium infection.

关 键 词：鸟分枝杆菌 重组PPE25蛋白 THP-1巨噬细胞 细胞凋亡 免疫应答 

分 类 号：R378[医药卫生—病原生物学]