摘要
目的研究结核分枝杆菌H37Rv重组热休克蛋白60(Heat shock protein 60 of Mycobacterium tuberculosis,MTB Hsp60)对巨噬细胞(RAW264.7)趋化功能的影响。方法MTB Hsp60重组蛋白(100 ng/mL)刺激巨噬细胞24 h后,采用实时荧光定量PCR检测下列趋化因子如单核细胞趋化蛋白1(Monocyte chemotactic protein-1,MCP-1,CCL2)、巨噬细胞炎症蛋白1α(Macrophage inflammatory protein-1α,MIP-1α,CCL3)、巨噬细胞炎症蛋白1β(Macrophage inflammatory protein-1β,MIP-1β,CCL4)、调节活化正常T细胞表达与分泌的趋化因子(Regulation of Activation,Expression and Secretion by Normal T Cells,RANTES,CCL5)和单核细胞趋化蛋白5(Monocyte chemotactic protein-5,MCP-5,CCL12)的mRNA表达;ELISA法检测细胞培养上清液中趋化因子CCL2、CCL3、CCL4、CCL5和CCL12的分泌水平;Western bolt检测巨噬细胞表面趋化因子受体CC基序趋化因子受体1(C-C motif chemokine receptor 1,CCR1)、CCR2和CCR5的蛋白表达;流式细胞术检测表达趋化因子受体CCR1、CCR2、CCR5的巨噬细胞;Transwell实验检测巨噬细胞的趋化功能。结果100 ng/mL MTB Hsp60重组蛋白刺激巨噬细胞24 h后,其趋化因子CCL2、CCL3、CCL4及CCL12的mRNA表达水平无明显差异,但CCL5的mRNA水平显著上调(P<0.05);巨噬细胞表面主要的趋化因子受体CCR1、CCR2蛋白表达水平显著增高(P<0.05),而其表面CCR5蛋白的变化不明显;流式细胞术检测结果显示,与未处理组相比,处理组中CCR1^(+)、CCR2^(+)巨噬细胞数量明显增加(P<0.05),而CCR5^(+)巨噬细胞数量无明显差异;Transwell实验表明,经过MTB Hsp60重组蛋白处理后的巨噬细胞有更明显的被募集的趋势(P<0.05);而上清液中趋化因子CCL2、CCL3、CCL4、CCL5及CCL12的释放水平无明显变化。结论MTB Hsp60重组蛋白能上调巨噬细胞中趋化因子CCL5的mRNA表达水平,能增加巨噬细胞表面趋化因子受体CCR1和CCR2的表达,并且能增强巨噬细胞的趋化功能。
Objective To study the effect of recombinant heat shock protein 60(MTB Hsp60)from Mycobacterium tuberculosis H37Rv on the chemotactic function of macrophages(RAW264.7).Methods After MTB Hsp60 recombinant protein(100 ng/mL)stimulated macrophages for 24 h,real-time fluorescence quantitative PCR was used to detect the mRNA expression of the following chemokines such as Monocyte chemotactic protein-1(MCP-1,CCL2),Macrophage inflammatory protein-1α(MIP-1α,CCL3),Macrophage inflammatory protein-1β(MIP-1β,CCL4),Regulation of Activation,Expression and Secretion by Normal T Cells(RANTES,CCL5),and Monocyte chemotactic protein-5(MCP-5,CCL12).The secretion levels of chemokines CCL2,CCL3,CCL4,CCL5 and CCL12 in cell culture supernatant were detected by ELISA;Western bolt assay was used to detect the expression of C-C motif chemokine receptor 1(CCR1),CCR2 and CCR5 on the surface of macrophages;Flow cytometry was performed to detect macrophages expressing chemokine receptors CCR1,CCR2,and CCR5;and Transwell assay was performed to detect the chemotactic function of macrophages.Results After MTB Hsp60 recombinant protein(100 ng/mL)stimulated macrophages for 24 h,the mRNA level of CCL5,not other chemokine(CCL2,CCL3,CCL4 or CCL12),was up-regulated significantly(P<0.05).However,the expressions of CCR1 and CCR2 proteins,not CCR5 protein,increased significantly(P<0.05).In addition,flow cytometry results showed that the number of CCR1^(+)and CCR2^(+)macrophages,not that of CCR5^(+)macrophages,in the stimulated groups increased significantly compared with that in the control groups(P<0.05),and Transwell assay results showed that macrophages treated with MTB Hsp60 recombinant protein had a more significant tendency to be recruited(P<0.05).Conclusion MTB Hsp60 recombinant protein upregulates the mRNA expression of chemokine CCL5 in macrophages,increases the levels of chemokine receptors CCR1 and CCR2 on the macrophage surface,and enhances the chemotactic function of macrophages.
作者
卫麟娜
高雪涵
董品志
吴荧浓
王海燕
罗军敏
覃明
WEI Linna;GAO Xuehan;DONG Pinzhi;WU Yingnong;WANG Haiyan;LUO Junmin;QINMing(Department of Immunology,School of Basic Medical Sciences,Zunyi Medical University,Zunyi 563000,Guizhou,China;Department of epidemiology and health statistics,School of public health,Zunyi Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2024年第2期157-161,171,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81860291)
2023年贵州省教学内容和课程体系改革项目(No.SJJG2023185)
作者简介
卫麟娜(1996-),女,陕西西安人,在读研究生,主要研究方向:感染免疫研究。E-mail:weilinna2021@163.com;通讯作者:覃明,E-mail:158277563@qq.com
