Immortalization of human dermal microvascular endothelial cells

9 Objectives: Microvascular endothelial cells (MECs) have been proved by increasing studies to play 10 important roles in the process of endocrine, immune response, and pathogenic microorganism infection. 11 However, most types of MECs have a limited number of divisions. Therefore, the immortalization of 12 primary MECs may provide a better cell model for research. And the present research is aimed to 13 establish an immortal human dermal microvascular endothelial cells (HDMECs). 14 Methods: To immortalize HDMECs, the telomerase reverse transcriptase (hTERT) gene was 15 transferred into the primary HDMECs by lentiviral infection. The passages of HDMECs transfected 16 with hTERT or without hTERT were analyzed. At the same time, the relative telomerase activity and 17 telomere length in HDMECs transfected with hTERT were detected by RT-PCR assay. And the 18 β-galactosidase (β-GAL) activity in HDMECs transfected with hTERT was detected by ELISA kits. 19 Finally, karyotype and tube formation analysis were used to evaluate the effects of transfection with 20 hTERT on the characteristics of HDMECs. 21 Results: The results showed that the number of passages of HDMECs transfected with hTERT was 22 significantly increased. The telomerase activity of HDMECs transfected with hTERT gene was 23 enhanced, and β-GAL activity was significantly reduced. Moreover, the transfection of hTERT gene 24 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. Karyotype analysis and tube formation analysis showed that there is no difference in the and relative length of chromosomes between hTERT-HDMECs and HDMECs, and they both have a pro-angiogenic phenotype that activates proliferation and migration. It shows that the characteristics of HDMECs transfected with hTERT have not changed. This is consistent with studies that the karyotype and tube formation of immortalized cells do not change and do not cause tumors, indicating that they can be safely used in future studies [23, 24] . In summary, transfection with hTERT can significantly increase the number of passages of HDMECs without changing the characteristics of endothelial cells.


29
MECs are distributed between capillaries and tissues, forming a barrier between blood vessels and tissues, which have various physiological functions such as regulating vasomotor, blood coagulation, 31 vascular permeability [1][2][3] . MECs are also involved in a series of physiological processes such as 32 regeneration, wound healing, inflammation, immune regulation and angiogenesis [4] . HDMECs has 33 become an important cell type in studies, and its division ability and activity directly affect the 34 accuracy of the experiment [5,6] . However, after multiple passages, the primary HDMECs will begin to 35 age or even stop proliferating. In order to ensure the number and stable state of HDMECs used for 36 research, we choose to immortalize HDMECs cultured in vitro.

37
Telomerase is a DNA polymerase that extends the 3' ends of chromosomes by synthesizing multiple 38 telomeric repeats [7] . It is a unique ribonucleoprotein (RNP) containing a specialized telomerase reverse 39 transcriptase (TERT) and telomerase RNA (TER) [8] . Telomerase is active in most human tumors but 40 not expressed in most non-immortalized somatic cells, which can inhibit telomere erosion and prevent 41 cell cycle senescence and apoptosis due to telomere length shorting [9] . At present, many cell lines have 42 been successfully established with transfected telomerase, which can maintain the basic physical and 43 chemical properties of primary cells [10,11]

HDMECs proliferation
To the 10th generation, HDMECs are obviously deformed, senile and dead (Fig. 4A). All of the 121 hTERT-HDMECs exhibited an elongated, "cobblestone like" shape, while maintaining their 122 proliferation activity even at high numbers of passages ( Fig. 4B-C). As shown in Fig. 4D  and they are no change in the cell characteristics [12] .

146
At present, most studies on endothelial cells use HDMECs or human umbilical vein endothelial cells 147 (HUVECs) [13] . Since HDMECs is prone to aging and has a limited number of divisions, it needs to be 148 purchased again or separated from the tissue [14] . In addition, if the source of ECs is different, the 149 physiological characteristics of ECs will be different, which is determined by the heterogeneity of endothelial cells [15] . In addition, spontaneous immortalization is rare in most cells. Therefore, the 151 immortalization of primary MECs is a way to solve the limitations of MECs.

Immortalizing cells by introducing foreign genes into cells through viruses is a commonly used method
153 of immortalization, such as simian virus 40 (SV40) or hTERT [16] . However, compared with primary 154 cells, SV40 transfection may induce cell chromosomal abnormalities, cell cycle control changes and 155 increased carcinogenic risk [17] . Some studies have confirmed that hTERT transfection does not cause 156 malignant transformation of cells, and does not change cell characteristics and karyotype stability [18,19] .

157
Previous studies have confirmed that cells can be immortalized by transfection with hTERT, which is 158 usually used to increase the possibility of cell immortalization and ultimately obtain an immortalized 159 cell line [20] . Therefore, in this experiment, we introduced hTERT into HDMECs through lentivirus

164
Cell senescence refers to the stable stagnation of cell proliferation. β-GAL activity is enhanced in 165 senescent cells, which means that increased β-GAL activity implies cell senescence [21] . Analysis of 166 β-GAL activity showed that HDMECs, but not hTERT-HDMECs, showed signs of aging. It can be 167 seen that HDMECs transfected with hTERT slowed down senescence and maintained the ability of cell 168 division and proliferation.

169
Telomerase is an enzyme responsible for maintaining the length of telomeres. Since telomerase is 170 inactivated or not expressed in most somatic cells, the replication potential of the cell is limited [13] .

171
When telomeres are shortened, the cell cycle is inhibited. In this study, we observed an increase in 172 telomere activity in HDMMCs transfected with hTERT compared with HDMECs. However, 173 transfection of hTERT did not lead to telomere lengthening of HDMMCs. Studies have shown that the 174 space of the longer telomeres in the immortalized cell nucleus overlaps, even if the same intensity 175 value is maintained, the volume of telomeres seems to be variable, which indicates that telomeres of 176 the same length can be more or less compact [22] . In our study, the telomere length in hTERT-HDMECs 177 was shorter than that in HDMECs. This may be because hTERT-HDMECs can maintain division and 178 proliferation through the spatial overlap of longer telomeres. hTERT may only extend the shortest 179 telomere length, rather than all telomere lengths.
Karyotype analysis and tube formation analysis showed that there is no difference in the number and 181 relative length of chromosomes between hTERT-HDMECs and HDMECs, and they both have a 182 pro-angiogenic phenotype that activates proliferation and migration. It shows that the characteristics of 183 HDMECs transfected with hTERT have not changed. This is consistent with studies that the karyotype 184 and tube formation of immortalized cells do not change and do not cause tumors, indicating that they 185 can be safely used in future studies [23,24] . In summary, transfection with hTERT can significantly 186 increase the number of passages of HDMECs without changing the characteristics of endothelial cells.             The β-GAL viability of HDMECs.