Establishment And Characteristic Of Immortalised Swine Umbilical Vein Endothelial Cell Line

Immortalised cell lines provide more advantages than primary cells, such as better uniformity of different cultures, better availability and easier genetic manipulation. Most research groups used primary isolated cells for their studies, accepting time- and cost-effective preparation steps and common variations in the cell's physiology. These facts make it difficult to compare results from experiments with different cell preparations or passages. Many studies showed temolerase activity in cells is transcription control of TERT that act as a reverse transcriptase. Introduction of hTERT could enhance the activity of temolerase in normal cells, elongate the life span of various cells and even result in immortalisation. hTERT transfected cells had no cell cycle runaway, karyotype instability, loss of serum dependent,which are neoplastic transformation feature..Swine endothelial cells are commonly used and well characterized in vitro model for studying blood-brain barrier features and some hemorrhagic disease. However, primary culture of swine cells have a finite proliferative lifespan before they undergo permanent growth arrest, known as replicative senescence, the inability to generate large amounts of cells, and phenotypical instability. To overcome these problems, working out a reliable system for further studies, it was necessary to establish a homogenous cell population with expanded lifespan, consistent for endothelial functions and characteristics. In this study, we isolated swine umbilical vein endothelial cells(SUVECs)in vitro, the plasmid pCI-neo-hTERT was transfected into SUVECs by lipofection. Experimental results obtained as follows:1. Swine umbilicus veins were isolated. 1.0 g/L callagenase I was poured into umbilicus vein and digested. Endothelial cells obtained was collected by centrifugation, cultured in M199 + 20% fetal calf serum at 37℃and 5% CO2. The result showed a confluent monolayer was former by 6 days, the endothelial cells looked like cobblestones and were stained positively for factorⅧrelative antigen , whicht shows cells cultured are endothelial cells. In the culture of 10 passages, cells entered into the senescence phase, finally the population appears growth retardation, and cells can not subculture again.2. swine umbilical vein endothelial cells were transfected with pCI-neo-hTERT by lipofectamine. 48 h after transfection, cells were screened with 500μg/ml G418 for 14 days. Drug resistant cells were selected and matained in 250μg/ml to ensure a stably positive population, positive clones were expanded for further culture. The transfected cells were detected the expression of hTERT mRNA by RT-PCR. The results showed that the hTERT mRNA was expressed in transfected cells, the ectogenic gene hTERT has integrated into genome of endothelial cells stably. The telomerase activity of the transfected cells were detected by TRAP-ELISA in 10 passages, 20 passages and 30 passages. The results showed the activity of telomerase was positive in transfected cells, while it was negative in SUVECs without transfection. The activity of telomerase in SUVECs can be activated through ectogenic gene hTERT transduction. One of clones, c2, was cultured up to 56 passages after introduction of the hTERT. We named it as immortalised swine umbilical vein endothelial cells line with hTERT (hTERT-SUVECs).3. During continuous culturing in vitro, endothelial cells was fusiform, and grew as confluent monolayer with the cobblestone morphology. The cells maintained contact inhibition, had no phenomenon of heteromorphous change and inverted volume ratio between nucleus and cytoplasm. The transfected cells were stained positively for F-ⅧRAg and CD34 factor, it showed phenotype antigen of cells had no loss. Through map of growth curve and observation of cell culture, we find that the transferred cells have the normal cell generation cycle and contact inhibition. The cell cycles and cell apoptosis of hTERT-SUVECs and SUVECs were detected by FCM, the results showed hTERT-SUVECs had higher proliferation activivity, and the ratio of apoptosis cells in hTERT-SUVECs was far lower than it in SUVECs. The above results showed that the immortalised cells maintained the properties of normal cells.4. Karyotype analysis revealed that hTERT-SUVECs had a normal modal chromosome; both cells have a near-diploid karyotype, with a mode chromosome number of 38. hTERT-SUVECs were injected into nude mice, after 2 months, no tumors developed in nude mice. The ability of hTERT-SUVECs and SUVECs to grow in anchorage-independent manner by using a clonogenic soft agar assay. The result showed that hTERT-SUVECs failed to form any colonies after 2 weeks when grown in soft agar. The proliferative ability of hTERT-SUVECs cultured in medium containing different concentration serum was detected by MTT assay. The result showed that the proliferative ability of hTERT-SUVECs cultured in medium containing high concentration of serum is higher than it in low concentration of serum. The serum requirement of the immortalised cells was not lose. Above results indicated that hTERT-SUVECs maintained the propertied of normal cells, and had no neoplastic transformation.5. PGI2 and ET-1 levels of culture supernate in hTERT-SUVECs and SUVECs were measured by radio-immunoassay (RIA), NO levels were measured by nitrate reductase assay. The results showed that secretory volumes changes of three vasoactive substances in both cells were similar at a cell proliferation period, the levels of PGI2 and ET-1 at same culturing time interval with same inoculative concentration were closed in both cells. But NO content in hTERT-SUVECs was lower than SUVECs in general. However, it could not present that the secretion of the hTERT-SUVECs was loss. These results indicated that biologic functions and metabolic capacities of hTERT -SUVECs was approximate to untransfected SUVECs. The introduction of hTERT gene was not change endothelial cells physiological functions.Overall results suggested that the hTERT was transfected into SUVECs successfully, the cell proliferation life-span was elongated in vitro, cells immortalised by hTERT retain their original characteristic. Therefore we concluded that the immortalised SUVECs were established. It was first reported in domestic and foreigh.