创伤弧菌溶细胞素融合蛋白细胞毒活性相关分子机制研究  

Study on WC recombinant protein cytolytic activity mechanism of an extracellular cytolytic toxin of Vibrio vulnificus

作　　者：桂静[1] 胡蝶[1] 郑丽[1] 楼永良[1] 肖美英[1] 严杰[2] 朱晔晶[1] 肖翔[1] 

机构地区：[1]温州医学院微生态学研究所,浙江省医学遗传学重点实验室,325035 [2]浙江大学医学院病原生物学教研室

出　　处：《中华微生物学和免疫学杂志》2008年第5期390-394,共5页Chinese Journal of Microbiology and Immunology

基　　金：浙江省自然科学基金（X205004）

摘　　要：目的研究创伤弧菌溶细胞素融合蛋白（rVVC）诱导人ECV304细胞（人脐静脉内皮细胞）凋亡过程中caspase．3，-8，-9活性变化。方法应用MTT法、Hoehest33342／PI荧光双染、流式细胞术及DNA琼脂糖凝胶电泳分析rVVC对人ECV304细胞诱导凋亡的影响；比色法测定rVVC诱导人ECV304凋亡过程中caspase-3，-8，-9活性变化。结果MTT结果显示rVVC具有降低人ECV304细胞的存活率活性；浓度为2．0溶血单位（HU）／ml的rVVC作用人ECV30412h后，其诱导凋亡的活性高于对照组和浓度为0．5HU／ml的rVVC处理组，具有剂量依赖性；浓度为2．0HU／ml rVVC处理组加40μmol／L Caspase全酶抑制剂（Z—VAD—FMK）后凋亡率较2．0HU／ml rVVC处理组有一定程度降低。浓度为2．0HU／ml rVVC处理人ECV304细胞0．5h后caspase-3活性开始增高，于3h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rVVC对人ECV304具有凋亡诱导的生物学活性，caspase-3可能与活性rVVC诱导的人ECV304凋亡有关。Objective To investigate the cytolytic activity of extracellular cytolytic toxin rVVC of Vibrio vulnificus on the apoptosis of human ECV304 cells, and to analyze the activities of Caspase-3 ,-8 and - 9. Methods The cytotoxic effect of refolded rVVC on the growth and apoptosis of ECV304 cells was identified by MTY, Hocbest33342/PI fluorescent staining, flow cytometry and DNA agarose electrophoresis analysis, respectively. The activities of Caspase-3, -8 and -9 was measured using a colorimetric method. Results The viability of human ECV304 cells exposed to rVVC was inhibited by rVVC after 24 h. 2.0 HU/ml rVVC groups had a better cytotoxic effect to human ECV304 than that of 0.5 HU/ml rVVC groups. The apoptosis of human ECV304 cells in 2.0 HU/ml rVVC ＋ 40 μmol/L Z-VAD-FMK groups was relative reduced than that of 2.0 HU/ml of rVVC groups. After 0.5 h treatment with 2.0 HU/ml of rVVC, the Caspase-3 activity in human ECV304 cells increased gradually and reached the peak at 3 h （ versus control groups, P 〈0.01 ）. The activity of Caspase-8 and -9 remained unchanging. Conclusion The rVVC has cytotoxic effect on human ECV304 and the cytolysin is probably correlated with Caspase-3.

关 键 词：创伤弧菌 溶细胞素 rVVC 人ECV304 细胞凋亡 

分 类 号：R378.3[医药卫生—病原生物学]