人巨细胞病毒临床分离株感染血管内皮细胞伴肾素基因表达

Renin gene expression in the vascular endothelial cells infected by the clinical isolate of cytomegalovirus

目的探讨巨细胞病毒(Cytomegalovirus,CMV)是否感染血管内皮细胞伴肾素表达。方法(1)用107pfu(空斑形成单位)/ml CMV临床分离株BI-5和实验室型CMV AD169分别与106腹主动脉内皮细胞和人脐静脉内皮细胞共同孵育,在23小时后、第3天、第7天、第10天和第14天分别收集培养上清200μl,第14天用PBS缓冲液洗细胞3次,收获细胞。每组实验均设培养液代替病毒液的无感染对照;(2)COBAS定量PCR检测培养上清中CMV DNA拷贝数;(3)PCR检测感染细胞中CMVpol基因;(4)RT-PCR、Real time RT-PCR和Western blot检测肾素在感染细胞内的表达。结果(1)BI-5和AD169感染静脉和动脉细胞后,其形态学变化相似,无细胞裂解病理效应;(2)AD169感染细胞不同时间培养上清中CMV DNA拷贝数无明显增加,BI-5呈增殖趋势;(3)BI-5感染动脉细胞CMV DNA拷贝数和肾素表达量均大于静脉细胞。结论临床分离株CMV以非裂解形式在血管内皮细胞持续存在并诱导肾素基因表达,血管内皮细胞分泌肾素可能是CMV感染引起心血管疾病的新机制。

Objective To explore whether or not cytomegalovirus （CMV） infected cardiovascular endothelial cells with renin gene expression. Methods （ 1 ） Umbilical vein cell line and abdominal aorta cell line were respectively infected with the clinical isolate of CMV BI-5 and the laboratory strain of CMV AD169 at multiplicities of infection （MOI） of 10 of each virus （ 107 pfu/ml CMV/106 ceils） and mock infected with medium alone. The supernatant of 200μl from each culture was collected at the 23rd hour, day 3, 7, 10, and 14 post-infection and replaced back with 200μl of fresh medium. At the 14th day of post-infection, the cells were rinsed with PBS three times and then harvested ; （2） CMV DNA copies in the supernatant from each cuhure were determined by COBAS PCR;（3） CMV DNA polymerase （pol） gene in the infected cells was tested by PCR; （4） The renin expression in the viral infected cells was measured with RT-PCR,real time quantity RT-PCT and western blot. Results （ 1 ） Lytic replication of CMV was not found in both venous and arterial cells infected till the 14th post-infective day; （2） The clinical isolates BI-5 persistently replicated in both venous and arterial endothelial cells and their supernatants but not the AD169, determined by viral pol gene copy number in both types of cells infected; （3） CMV DNA and renin expression in CMV infected arterial cells both were slronger than in venous cells. Conclusiou CMV was nonlytic and continuously released from the infected vascular cells for the life span of the culture, and induced renin expression in CMV-infected cells. The locat renin from CMV- infected cells possibly plays an important role in pathogenesis of atherosclerosis.