rVvhA诱导小鼠单核巨噬细胞凋亡的作用及其机制  

Effect of apoptosis of rVvhA on J774A. 1 and the related mechanism

作　　者：王波[1] 金疆[1] 钟毓红[1] 赵志超[1] 杨军[1] 楼永良[1] 严杰[2] 

机构地区：[1]温州医学院检验医学院,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》2010年第3期239-244,共6页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：浙江省自然科学资金（Y2090468）;浙江省科技计划（84008012）

摘　　要：目的研究重组创伤弧菌溶细胞素（rVvhA）诱导小鼠单核巨噬细胞（J774A．1）凋亡的作用及机制。方法MTT法、电子透射电镜、流式细胞仪结合AnnexinV-PI标记法、线粒体膜电位和caspase活性检测等方法测定rVvhA诱导J774A．1凋亡的作用。结果MTT结果显示rVvhA能够抑制J774A．1生长。2．0溶血单位（HU）／ml和3．0HU／ml的rVvhA作用J774A．18h后，透射电镜观察细胞和线粒体形态发生凋亡改变；流式细胞仪检测凋亡率分别为（7．80±0．62）％、（12．33±0．12）％，均高于正常组（3．07±0．67）％；线粒体膜电位也下降，流式细胞仪结果显示绿色荧光率分别是9．8％、39．2％。其中3．0HU／mlrVvhA作用组，caspase-3、9活性增加，并且具有时间依赖性，而caspase-8活性没有明显改变。3．0HU／mlrVvhA加caspase-3抑制剂（Ac—DEVD-FMK）或caspase-9抑制剂（Ac—LEHD-FMK）的凋亡率与3．0HU／mlrVvhA作用组相比都降低，分别为（6．23±3．95）％、（9．60±3．14）％；同时caspase-3、9活性也下降。结论rVvhA具有诱导J774A．1凋亡的生物学活性；其机制可能与依赖caspase的线粒体途径有关。Objective To investigate the activity of recombinant Vibrio vulnificus hemolysin （rVvhA） on the apoptosis of J774A. 1 cells and the related mechanism. Methods The cytotoxic effect of rVvhA on the growth of J774A. 1 cells was identified by MTT, celluar and mitochondrial morphology were observed by transmission electron microscopy, apoptosis or necrosis and mitochondrial membrane potential in J774A. 1 cells were measured by flow cytometry, activities of caspase-3 ,-8 ,-9 were detected by spectropho- tometry. Results The viability of J774A. 1 cells exposed to rVvhA was inhibited, and it is dependent on dose. Celluar and mitochondrial ultrastructure both occurred to change obviously observed by transmission electron microscopy in J774A. 1 treated by 2.0 HU/ml and 3.0 HU/ml rVvhA after 8 hours; and 3.0 HU/ml rVvhA group had a better cytotoxic effect on J774A. 1 than that of 3.0 HU/ml rVvhA group. The percentage of apoptosis is （7.80 ±0.62）%, （12.33 ± 0. 12）%, respectively. Besides, the mitochondrial membrane potential also reduced, because the rate of fluorescence which is green increase 1.0% （normal） to 9.8% （2.0 HU/ml rVvhA） and 39.2% （3.0 HU/ml rVvhA）. At the same time, the caspase-3, -9 activity increased gradually, but caspase-8 remained unchanging. In J774A. 1 cells treated by 3.0 HU/ml rVvhA ＋ caspase-5 inhibitor（Ac-DEVD-FMK） or caspase-9 inhibitor（ Ac-LEHD-FMK）, The apoptosis of was reduced to （6.23 ± 3.95 ） %, （9.60 ±3.14） %, and the activity of caspase-3, -9 reduced, too. Conclusion The rVvhA has cytotoxic effect on J774A. 1. Mitochondria-mediated apoptosis pathway which is dependent on caspase may be related to apoptosis induced by rVvhA in J774A. 1.

关 键 词：创伤弧菌 溶细胞素 J774A.1 凋亡 线粒体 

分 类 号：R363[医药卫生—病理学]