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| TcpC induces apoptosis of human vascular endothelial cells and its mechanisms |
| ZHANG Chong1, ZHOU Jia-Le1, FANG Jie1, ZHANG Da-Yong1, WANG Bao-Ming1, CHEN Rui-Ling1,2, PAN Jian-Ping1 |
| 1.Department of Medicine,Zhejiang University City College School of Medicine，Hangzhou 310015，China; 2.Department of Pathogen Biology，Zhejiang University School of Medicine，Hangzhou 310058，China; |
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Abstract Objective： To investigate the effects of TcpC on human umbilical vascular endothelial cells (HUVECs) and its mechanisms.
Methods： HUVECs were co-cultured with TcpC secreting wild-type E. coli strain CFT073 (TcpCwt) or tcpc gene-deleted CFT073 mutant strain (TcpCmut) in transwell system，respectively. Apoptosis of HUVECs was analyzed by Annexin-V/PI double staining. Mitochondrial membrane depolarization was detected by JC-1 staining. Expression of apoptosis-related proteins in HUVECs was determined by Western blot.
Results： HUVECs showed morphological changes after co-cultured with TcpCwt for 24 h：the cells became detached and cell debris increased，and cell number was also decreased when compared to HUVECs co-cultured with TcpCmut. The apoptosis of HUVEC cells co-cultured with TcpCwt for 24 h significantly increased，compared to that of control group and TcpCmut group (60.1% 9.7% vs 9.0% 1.3% and 16.9% 0.4%，respectively, P<0.05); meanwhile the mitochondrial depolarization of HUVECs co-cultured with TcpCwt was significantly increased，compared to that in control and TcpCmut groups (64.5% 0.9% vs 14.5% 2.1% and 15.6% 3.3%，respectively，P<0.05). Cleavage of PARP and inhibition of Mcl-1 and XIAP expression were seen in HUVECs co-cultured with TcpCwt，but not in groups of control and TcpCmut.
Conclusion： TcpC secreted from CFT073 can induce apoptosis of HUVECs through mitochondrial pathway，in which PARP is cleaved and Mcl-1 and XIAP expressions are inhibited.
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Received: 14 June 2013
Published: 25 September 2013
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TcpC诱导人血管内皮细胞凋亡及其机制
目的：研究TcpC对人血管内皮细胞凋亡的影响及其机制，为阐明大肠埃希菌所致败血症的病理生理机制提供新的实验依据。
方法：以人脐静脉内皮细胞作为研究对象，将表达TcpC的CFT073野生株（TcpCwt）和敲除tcpc基因的CFT073突变株（TcpCmut）与人脐静脉内皮细胞共培养；Annexin V/PI双染法检测细胞凋亡；JC-1染色法检测线粒体膜电位变化；Western blotting检测凋亡相关蛋白的表达。
结果：TcpCwt与人脐静脉内皮细胞共培养24 h后，人脐静脉内皮细胞数量较少，有脱壁现象，且多数细胞呈碎片状。与TcpCmut相比，TcpCwt能显著诱导人脐静脉内皮细胞发生凋亡（P<0.05）。TcpCwt能显著降低人脐静脉内皮细胞线粒体膜电位，而TcpCmut处理的人脐静脉内皮细胞线粒体膜电位变化与空白对照差异无显著性意义。TcpC能增加多聚ADP核糖聚合酶蛋白裂解，抑制抗凋亡蛋白Mcl-1及XIAP的表达。
结论：大肠埃希菌分泌的TcpC能通过线粒体途径引起多聚ADP核糖聚合酶切割，并抑制抑凋亡蛋白Mcl-1和XIAP的表达，诱导血管内皮细胞凋亡。
关键词：
肠致病性大肠杆菌,
内皮,
血管,
内皮细胞,
脐静脉,
线粒体,
细胞凋亡,
小鼠,
基因敲除,
细胞,
培养的
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