人脐静脉内皮细胞永生化及生物学特性的研究  

Immortalization of human umbilical vein endothelial cells and the effect of immortalization on its biological characteristics

作　　者：李盛波 郑勇斌[2] Li Shengbo;Zheng Yongbin(Department of Critical Care Medicine,Renmin Hospital of Wuhan University,Wuhan 430060,China;Department of Gastrointestinal Surgery,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院重症医学科,430060 [2]武汉大学人民医院胃肠外科,430060

出　　处：《中华实验外科杂志》2019年第1期60-62,共3页Chinese Journal of Experimental Surgery

摘　　要：目的建立永生化人脐静脉内皮细胞(HUVECs)系并探讨永生化对HUVECs增殖、凋亡及成瘤性影响。方法扩增并提取猿猴病毒40大T抗原(SV40LT)和人端粒酶反转录酶催化亚基(hTERT)基因,反转录病毒载体介导感染HUVECs;筛选2周;聚合酶链反应(PCR)扩增后行琼脂糖凝胶电泳成像鉴定目的基因是否转入HUVECs;细胞计数法比较细胞增殖、流式细胞术检测细胞凋亡、裸鼠成瘤实验检测细胞恶性转化。 结果筛选出抗嘌呤霉素阳性克隆,SV40LT、hTERT成功转入HUVECs;永生化组细胞生长曲线上移,细胞增殖第5、6、7天,SV40LT永生化组及hTERT永生化组与HUVECs组细胞数目差异有统计学意义(P<0.05),hTERT永生化组和HUVECs组细胞数目差异有统计学意义(P<0.05);HUVECs组细胞总凋亡率为(11.2±0.9)%,SV40LT永生化组HUVECs总凋亡率为(3.4±0.4)%,hTERT永生化组HUVECs总凋亡率为(2.8±0.4)%,SV40LT永生化组、hTERT永生化组凋亡率均低于HUVECs组(均P<0.05);两种永生化细胞株不具成瘤性。结论SV40LT、hTERT基因能使HUVECs发生永生化,永生化HUVECs增殖能力增强,凋亡率下降,无恶性转化。Objective To establish the immortalized human umbilical vein endothelial cell lines and investigate the effect of immortalization on biological characteristics of proliferation, apoptosis and tumorigenesis of the cells.MethodsPlasmid containing simian virus 40 large T antigen (SV40LT) gene or human telomerase reverse transcriptase (hTERT) gene was amplified and extracted;the target genes marked by puromycin resistance were packed by human embryo kidney cells (293T) to get the retroviral vectors and used to infect human umbilical vein endothelial cells (HUVECs);then the infected cells were screened by culture medium containing 2 μg/ml puromycin for two weeks;and polymerase chain reaction (PCR) was used to identify whether the target genes were successfully transferred into HUVECs;cell counting method, flow cytometry and tumorigenesis experiments in nude mice were used to detect the biological characteristics of proliferation, apoptosis and malignant transformation of immortalized cells respectively.Results The human umbilical vein endothelial cells were successfully immortalized by retrovirus-mediated SV40LT and hTERT;On the 5th to the 7th day, there were significant differences in cells number between the SV40LT immortalized group and HUVECs group (P<0.05) and between the hTERT immortalized group and HUVECs group (P<0.05). The apoptosis rate of HUVECs group was (11.2±0.9)%, the apoptosis rate of HUVECs immortalized by SV40LT was (3.4±0.4)%, the apoptosis rate of HUVECs immortalized by hTERT was (2.8±0.4)%. The apoptotic rates of the two immortalized group were lower than that of HUVECs group (both P<0.01). These immortalized HUVECs had no tumorigenicity.Conclusion Retrovirus-mediated SV40LT and hTERT can successfully immortalize HUVECs;the proliferative capacity of immortalized HUVECs is greatly enhanced;the apoptosis rate is decreased, and no malignant transformation occurs.

关 键 词：猿猴病毒40大T抗原 人端粒酶反转录酶催化亚基 反转录病毒载体 人脐 静脉内皮细胞 永生化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]