创伤弧菌溶细胞素体外诱导人脐静脉内皮细胞凋亡及其在细胞中的定位  

Apoptosis-inducing effect and intracellular location of Vibrio vulnificus cytolysin to human umbilical vein endothelial cell in vitro

作　　者：赵金方[1] 丁威[2] 赵旭鸿[1] 陆淼泉[1] 严杰[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310058 [2]温州医学院检验医学院

出　　处：《中华微生物学和免疫学杂志》2008年第11期976-981,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的了解创伤弧菌溶细胞素（Vibrio vulnificus cytolysin，VVC）诱导人脐静脉内皮细胞（HUVEC）凋亡作用及其机制。方法采用PCR扩增创伤弧菌GTC333株VVC编码基因whA，T—A克隆后测序并构建whA基因原核表达系统E．coli BL21DE3^pET-42a-vvhA。采用Ni—NTA亲和层析法提纯目的重组蛋白rVVC，SDS-PAGE联合Bio—Rad凝胶图像分析系统确定rVVC表达量及提取物纯度。采用溶血试验检测rVVC溶解兔红细胞的活性。2，4-二硝基苯肼比色法和四苯硼钠比色法分别测定rVVC作用的HUVEC培养物上清中乳酸脱氢酶（LDH）和K^＋含量。采用流式细胞术检测rVVC诱导HUVEC凋亡的作用。将FITC标记rVVC，采用激光共聚焦技术对HUVEC中的FITC标记rVVC进行定位。结果与GenBank中相关序列比较，所克隆的whA基因核苷酸和氨基酸序列相似性分别为96．09％和98．26％。1μg／ml的rVVC即可溶解兔红细胞（P〈0．01）。10μg／ml的rVVC可引起HUVEC培养物上清中K^＋含量明显增高（P〈0．01），但LDH含量无明显改变（P〉0．05）。1～100μg／ml的rVVC作用2h可使HUVEC凋亡。在FITC标记rVVC作用HUVEC5—240min内，rVVC逐渐从细胞膜表面向膜内侧和胞浆内移行，作用30min时多数rVVC进入细胞。结论rVVC具有溶细胞素活性。VVC可进入HUVEC，诱导细胞凋亡是其损伤HUVEC的主要机制。Objective To determine the effect of Vibrio vulnificus cytolysin （VVC） inducing apoptosis in human umbilical vein endothelial cell（HUVEC） and its possible mechanism. Methods The entire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced after T-A cloning. E. coli BL21DE3^pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then constructed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K^＋ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apoptosis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labeling rVVC in HUVEC was observed by laser confocal microscopy. Results The cloned vvhA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding sequences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes （P 〈0.01 ）. 10 μg/ml rVVC was able to promote the increases of K^＋ content （P 〈 0.01 ） but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1 -- 100μg/ml of rVVC for 2 h. In the 5 - 240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rVVC gradually moved from surface to inner side of the membrane and then entered the cytoplasma. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.

关 键 词：创伤弧菌 溶细胞素 重组表达 人脐静脉内皮细胞 凋亡 

分 类 号：R378[医药卫生—病原生物学]