Immortalization of human dermal microvascular endothelial cells

Abstract

Objectives : Microvascular endothelial cells (MECs) have been proved by increasing studies to play important roles in the process of endocrine, immune response, and pathogenic microorganism infection. However, most types of MECs have a limited number of divisions. Therefore, the immortalization of primary MECs may provide a better cell model for research. And the present research is aimed to establish an immortal human dermal microvascular endothelial cells (HDMECs). Methods : To immortalize HDMECs, the telomerase reverse transcriptase (hTERT) gene was transferred into the primary HDMECs by lentiviral infection. The passages of HDMECs transfected with hTERT or without hTERT were analyzed. At the same time, the relative telomerase activity and telomere length in HDMECs transfected with hTERT were detected by RT-PCR assay. And the β-galactosidase (β-GAL) activity in HDMECs transfected with hTERT was detected by ELISA kits. Finally, karyotype and tube formation analysis were used to evaluate the effects of transfection with hTERT on the characteristics of HDMECs. Results: The results showed that the number of passages of HDMECs transfected with hTERT was significantly increased. The telomerase activity of HDMECs transfected with hTERT gene was enhanced, and β-GAL activity was significantly reduced. Moreover, the transfection of hTERT gene has almost no effect on the karyotype and tube formation of HDMECs. Conclusion: These data indicate that transfection of hTERT gene could successfully enhance the cleavage ability of HDMECs, and the characteristics of hTERT-HDMECs remain almost unchanged.