Study On Immortalization Of Swine Umbilicus Veins Endothelial Cells Transfected Telomerase Reverse Transcriptase Gene

CSFV could replicate and proliferate in multiple animals'tissue cells, but not cause the cytopathic effect (CPE) in general condition.Our research found pathogenic swine appearing degeneration and necrosis of endothelial cells of capillary and small vascular after inoculation of CSFV,for the purpose of facilitating the study on pathologic mechanism of CSFV and offering opptisum cell model,we establish in vitro culture method of swine Umbilicus veins endothelial cells. However, the cells for basic research and therapeutic purposes has been restricted partly by their limited proliferative potential which they could not subculture more than ten passages becoming senescent and dead.Several immortalized cell lines have been established either spontaneously or via gene (including SV-40 large T antigen and HPV 18 E6/E7) transduction. As the proteins encoded by the virus disrupt many cellular pathways, transformed cells tend to be genetically unstable, including polynucleation, and loss and gaining whole or partial chromsomes. Moreover, some undesirable changes, such as loss of some differentiated properties and normal cell-cycle checkpoint controls immerged inevitably in these cells.In order to overcome the above shortcomings,human telomerase reverse transcriptase (hTERT) gene was transfected into primary swine umbilicus veins endothelial cells. Subsequent activation of telomerase allowed the cells to become immortal.1. Swine umbilicus veins were washed by PBS. 0.1% callagenase I was poured into umbilicus vein and digested. Primary swine umbilicus veins endothelial cells were transfected with eukaryotic expressing plasmid pCIneo-hTERT encoding hTERT and neo genes. After selection with G418 for a month, anti-G418 positive cell clones were appeared and positive cell clones were selected using filter paper and expanded for continuous culture. After stable transfection, telomerase activity turned to be positive in these transfected cells. Cells has been maintained in culture for about 2 months, up to 15 passages till now.2. The protein and phenotypical changes of the transformants were examined. The result showed that the plating efficiency to plastic was 70% for 24h in coolagenase dispersed cells.A confluent monolayer was former by 3～4 days. The endothelial cells looked like cobblestones.