Journal of Zhejiang University SCIENCE
(ISSN 1009-3095, Monthly)

2005   Vol. 6B   No. 7   p.631-636

            [ Home Page ] | [ PDF Full Text ]   On-line Access Date:   June 23, 2005

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

BIAN Chang1, ZHAO Kui2, TONG Guo-xin3, ZHU Yong-liang1, CHEN Peng†1

(1Department of Cardiology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China)
(2Department of Nuclear Medicine, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310001, China)
(3Department of Cardiology, Hangzhou First Hospital, Hangzhou 310009, China)
 †E-mail: skyoutwin@yahoo.com.cn
Received Sept. 6, 2004; revision accepted Jan. 27, 2005

Abstract: Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods: Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination. Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

Key words: Endothelial cell, Telomerase activity, Immortalization
doi:10.1631/jzus.2005.B0631             CLC number: R543.6