创伤弧菌溶细胞素vvhA融合蛋白细胞毒活性相关分子机制的探讨  

Study on vvhA recombinant protein cytolytic activity mechanism of an extracellular cytolytic toxin of V. vulnificus

作　　者：桂静[1] 朱晔晶[1] 楼永良[1] 

机构地区：[1]温州医学院检验医学院

出　　处：《检验医学教育》2010年第2期43-48,共6页

基　　金：浙江省自然科学基金(YZ090468)

摘　　要：目的:研究创伤弧菌溶细胞素vvhA融合蛋白(rVvhA)诱导人脐静脉内皮细胞(ECV304)凋亡过程中caspase-3,-8,-9活性变化。方法:应用MTT法、Hochest33342/pI荧光双染、流式细胞术及DNA琼脂糖凝胶电泳分析rVvhA对人ECV304细胞诱导凋亡的影响;比色法测定rVvhA诱导人ECV304凋亡过程中caspase-3,-8,-9活性变化。结果:MTT结果显示rVvhA具有降低人ECV304细胞的存活率活性;浓度为2.0HU/ml的rVvhA作用人ECV304,12小时后,其诱导凋亡的活性高于对照组和浓度为0.5HU/ml的rVvhA处理组,具有剂量依赖性;浓度为2.0HU/mlrVvhA处理组加40μMcaspase全酶抑制剂(Z-VAD-FMK)后凋亡率较2.0HU/mlrVvhA处理组有一定程度降低。浓度为2.0HU/mlrVvhA处理人ECV304细胞30分钟后caspase-3活性开始增高,于3小时达高峰,与对照组比较差异有统计学意义(P<0.01),caspase-8,-9活性无明显变化。结论:rVvhA对人ECV304具有凋亡诱导的生物学活性,caspase-3可能与活性rVvhA诱导的人ECV304凋亡有关。Objective：To investigate the cytolytic activity of extracellular cytolytic toxin rVvhA of V. vulnificus on the apoptosis of human ECV304 ceils and analyze the activities of caspase-3,-8 and -9. Methods： The cytotoxic effect of refolded rVvC on the growth and apoptosis of human ECV304 cells was identified by MTT, H33342/PI fluorescent staining, flow eytometry and DNA agarose electrophoresis analysis, respectively. The activities of caspase-3,-8and-9was measured using a colorimetric method. Results： The viability of human ECV304 ceils exposed to rVvhA was inhibited by rVvhA after 24 hours. 2. 0HU/ml rVvhA groups had a better cytotoxic effect to human ECV304 than that of 0. 5 HU /ml rVvhA groups ;The apoptosis of human ECV304 cells in 2.0 HU/ml rVvhA ＋40μM Z-VAD-FMK groups was relative reduced than that of 2.0 HU/ rVvhA groups. After 0.5 hour＇s treatment with 2.0 HU/ml rVvhA, the caspase-3 activity in human ECV304 cells increased gradually and reached the peak at 3 hours （versus control groups, P 〈0.01）. The activity of caspase-8and-9remained unchanging. Conclusion：The rVvhA has cytotoxie effect on human ECV304 cells and the cytolysin is probably correlated with easpase -3.

关 键 词：创伤弧菌 溶细胞素 rVvhA 人脐静脉内皮细胞 细胞凋亡 

分 类 号：R446.5[医药卫生—诊断学]