Establish The Cultivation Of Swine Vascular Endothelial Cell In Vitro And Study On The Growth Characteristics

The purposes of the present experiment are to study swine vascular endothelial cell (SVEC) in vitro and to establish foundation for farther researching the biological characters of SVEC infected by Classical Swine Fever Virus(CSFV) and the mechanisms of diseases such as Hog Cholera. SVEC were successfully separated by using type I collagenase and trypsin and cultured. Growth character of SVEC were studied through primary culture, passage, purification and growth curve and so on. Augmenting cell proliferation of insulin were also studied with dosage-response curve. The results as follows:1. The inner membrane of swine vascular digested by type I collagenase or trypsin can provide enough dispersed cells for primary culture. But the effect of type I collagenase is apparently better than that of trypsin and the damage to SVEC of typeI collagenase is less than that of trypsin. Twenty-four hours after seeding, the SVECs grow and form many islet-like cell clusters. By forty-eight hours after seeding, the cell clusters increase in size and can be divide into a central region with crowded endothelial cells and a peripheral region with fewer cells. Primary cells can coalesce and form incomplete monolayer in six to seven days.2. Vascular of piglet aged thirty to forty days dissociated ten minutes by type I collagenase can provide the most endothelial cells. Fibroblasts mixing in endothelial cells increase obviously when the time exceeds ten minutes.3. Antibiotic Trypsin Versen(ATV)combing with pipette is more appropriate to digestion and passage of SVEC compared with 0.25% trypsin.4. Fibroblasts sometimes mixing in the primary and the early passage of SVECs can be cleared away by using 0.25% lemon acid trypsin based on that fibroblast is more sensitive to lemon acid trypsin than that of endothelial cell.5.The culture cells are observed with microscopy phase contrast and the cells arrange in shape of typical "cobble-stone". Contact inhibition in existing among cells. Observed with scanning electron microscopy(SEM), the cells are polygonal in shape or unregular. Factor VI-related antigen antibody indirect fluorescence staining can examine the obvious yellow-green fluorescent light in plasma around nucleus. All those can prove the cultured cell are endothelial cell.6. M-199 is more appropriate as culture medium to SVEC compared with MEM.7. Adding insulin and hydrocotisone to cultivating liquid can evidently augment cells proliferation. Dosage-response curve shows that the optimum dose of insulin is 8ug/mL.8. From growth cure, we know the SVECs are in a lag phase in first one day after passage, then they enter logarithmic growth phase and can last two to three days. From the fifth day, they enter stagnate phage (plateau). They begin to fall and die from the eighth day.