Transfection of human umbilical vein endothelial cells with lentivirus containing enhanced green fluorescent protein / 中国组织工程研究

ABSTRACT

BACKGROUND:Human umbilical vein endothelial cells transfected with lentivirus containing enhanced green fluorescent protein can be easily traced. The optimal multiplicity of infection and time for producing strong fluorescence intensity can lay the foundation of tracing human umbilical vein endothelial cells in animal models. OBJECTIVE:To observe expression of lentivirus containing enhanced green fluorescent protein in human umbilical vein endothelial cells, and thereby to find a stable method to label human umbilical vein endothelial cells. METHODS:Using 0.1%col agenase perfusion digestion, we isolated human umbilical vein endothelial cells, which then were placed into a culture medium containing 20%fetal bovine serum and endothelial cellgrowth factor and observed under an inverted microscope. Fol owing digestion, centrifugation and suspension, the cells were counted and divided into four groups, 5.0×105 cells in each group. After cells were seededonto 24-wel plates, 10μL serum-free Dulbecco’s modified Eagle’s medium was added into the blank group, and lentiviruses containing enhanced green fluorescent protein were added into another three groups for celltransfection respectively at multiplicities of infection of 2, 3, 4. There were three dishes in each group. RESULTS AND CONCLUSION:After cultured for 5-7 days, isolated cells grew into a single layer and exhibited a cobblestone-like arrangement under a light microscope. In addition, factor VIII related antigen test was positive. A green fluorescence was visible at 24 hours of transfection, and peaked at 72 hours. Transfection efficiency was in a linear growth with the multiplicity of infection. Up to the 21st day of transfection, the green fluorescence was stil visible. After 0, 7, 14, 21 days of transfection, the number of human umbilical vein endothelial cells showed no difference between the transfection group with the multiplicity of infection=3 and blank group, suggesting the proliferative ability of cells has no changes after transfection with lentivirus containing enhanced green fluorescent protein. These findings indicate that the lentivirus containing enhanced green fluorescent protein can highly transfect human umbilical vein endothelial cells, and green fluorescent protein can sustainably express for 21 days but cannot impact the cellproliferation.