重组创伤弧菌溶细胞素诱导THP-1细胞损伤的机制研究  

Role of rVvhA in inducing THP-1 cells damage

作　　者：陆晓雅[1] 陈建林[1] 刘彪[1] 谢旦立[1] 楼永良[1] 

机构地区：[1]温州医科大学检验医学院,检验医学教育部重点实验室,325035

出　　处：《中华微生物学和免疫学杂志》2013年第10期761-765,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省自然科学基金( Y2090468)；浙江省临床检验诊断技术科技创新团队资助

摘　　要：目的：研究重组创伤弧菌溶细胞素（rVvhA）对人单核细胞白血病细胞（THP-1）的损伤机制及其钙离子通道的研究。方法采用倒置显微镜、CCK-8法、Fluo3/AM法、caspase 活性检测等方法测定rVvhA对THP-1细胞的影响，并研究胞外Ca2＋的内流途径。结果 rVvhA作用后细胞形态学变化明显，细胞皱缩、变形甚至裂解，且呈剂量依赖性。12μg/ml rVvhA 作用THP-1细胞1 h后胞外K＋浓度升高，作用6 h后胞外LDH浓度显著升高。 Verapamil、Mibefradil和SKF-96365均不能抑制rVvhA引起的Ca2＋内流。同时rVvhA可导致THP-1细胞的caspase-3和caspase-9活性升高，并呈时间依赖性。结论 rVvhA对THP-1细胞具有明显的损伤作用，且可能通过caspase-9/3途径诱导细胞凋亡，并能引起细胞内Ca2＋浓度升高，升高的Ca2＋可能通过细胞膜上形成的孔道被动入胞。Objective To investigate the role of recombinant Vibrio vulnificus cytolysin （rVvhA） in inducing THP-1 cells damage and study the pathway of associated calcium influx .Methods Inverted mi-croscope, CCK-8 cell proliferation kit, Fluo3/AM staining and caspase activity detection were performed to analyze the damage of THP-1 cells induced by rVvhA and the pathway of calcium influx .Results rVvhA had cytotoxic effects on THP-1 cells in a dose-dependent manner .The concentrations of extracellular K ＋and LDH were respectively up-regulated after 1 h and 6 h of 12 μg/ml rVvhA intervention .Verapamil , Mibe-fradil and SKF-96365 could not prevent the influx of free Ca 2＋induced by rVvhA .The activities of caspase-3 and caspase-9 were singanificantly enhanced by rVvhA in a time-dependant manner .Conclusion rVvhA can induce THP-1 cells damage through triggering extracellular calcium influx via porous channel on cell membrane.Moreover, rVvhA might induce THP-1 cell apoptosis through activating caspase-9/3-dependent pathway .

关 键 词：重组创伤弧菌溶细胞素 人单核细胞白血病细胞 细胞损伤 钙离子通道 

分 类 号：R378[医药卫生—病原生物学]