Study On Immortalization Of Swine Endothelial Cells By HTERT Or SV40LT

Vascular endothelial cells (EC) are organized as a thin layer on the interiorsurface of all vessesls and are known to function in a variety of immprtantphysiological processes. Swine EC are the major targets of Classical swine fevervirus (CSFV). Infection of EC by CSFV causes severe widespread haemorrhages inpigs and represents the major cause of pigs’ death. EC is a good model for the studyof pathogenesis of CSFV.Primary EC have a limited life span in culture. After about10passages, theywill undergo permanent growth arrest, known as replacative senescence. Frequentlyisolate fresh primary EC cultures is a tedious process and also leads to significantvariation from one preparation to another, can not keep the stability of cellularphysiological indexes. In order to have sufficient, consistant cell material throughoutthe research of pathogenesis of CSFV, immortalized swine EC lines are needed to beestablished.In this study, fresh umbilical cords were obtained from piglest at farrowing.Vein vessels were separated carefully and infused with0.1%collagenase I solution.After10-min incubation at37℃water bath, cells were collected and cultivated.Abundant swine umbilical vein endothelial cells (SUVEC) were isolated from in allmore than200umbilical vein vessels. They grew as a confluent monolayer withcobblestone morphology. They were von Willebrand Factor positive and coulduptake Dil-Acetylated Low Density Lipoprotein (Dil-Ac-LDL). These resultsdemonstrated the cell cultures were endothelial cells. Preliminary6passaggesSUVEC grew fast and could be used for researches. Then the proliferation speeddecreased eventually the cells entered senescence. They could be cultured about10passges in all.In order to increase gene integrate rate, retrovirus mediated gene transduction was used to transfer hTERT and SV40LT gene into SUVEC, respectively.Recombinant retroviral vector pBABE-neo-hTERT and pVSV-G plasmid werecotransfected into GP2-293cells to package pseudovirus containing hTERT and neogene. Recombinant retroviral vector pBABE-puro-SV40LT and pVSV-G plasmidwere cotransfected into GP2-293cells to package pseudoviruses containing SV40LTgene and puro gene. Pseudoviruses were collected and used to infect SUVEC.Positive clones were picked following the selection using G418and puromycinrespectively from hTERT and SV40LT transduced monolayers, followed bycontinous passage and morphorlogical and phenotypical characterization. Two stableswine endothelial cell lines, SUVEC-hT (carrying hTERT gene) and SUVEC-ST(carrying SV40LT gene) were generated.The SUVEC-hT cell line has been cultured for120passages. The SUVEC-STcell line has been cultured for115passages. They grew as a monolayer withcobblestone morphology, had high proliferation capacity. They grew faster thanSUVEC and did not show senescent phenotype. Stable expression of hTERT geneand SV40LT gene were detectd in SUVEC-hT cells and SUVEC-ST cells,respectively, by both RT-PCR and Western Blotting. SUVEC-hT cells expressed ECspecific CD31, CD34and von Willebrand factor; they could uptake Dil-Ac-LDL.SUVEC-ST cells expressed CD31and CD34molecules and could uptakeDil-Ac-LDL.Karyotype analysis revealed both SUVEC-hT and SUVEC-ST cell lines kept38chromosomes and normal karyotypes. They kept anchorage dependence, contactinhibition, could not grow in soft agar. Cell proliferative ability assessments shewthe two lines maintained serum dependence. Cells failed to proliferate in serum-freemedium and proliferate speed responded to serum in a dose-dependent fashion. Itreached maximal speed with10%serum, while higer serum concentrations andaddition of endothelial cell growth supplement could not promote cell growth anymore. CSFV infection assay indicated two EC lines retained the susceptivity to theinfection with CSFV.These results suggest hTERT and SV40LT gene were transduced into SUVECand have lead to two immortalized swine endothelial cell lines SUVEC-hT and SUVEC-ST. They retain important endothelial cell characteristics, are normal cellsinstead of transformed cells. They will be useful to gain insight into the pathogenesisof viral haemorrhagic diseases such as CSFV.