[Transfection of human umbilical vein endothelial cell line ECV-304 with liposome-oligonucleotide complexes under hypothermic and anoxic conditions].
OBJECTIVE
To investigate the transfection efficiency of nuclear factor (NF)-kappaB decoy oligodeoxynucleotides (ODN) mediated by in vivo liposome in human umbilical vascular endothelial cell line ECV-304 under hypothermic and anoxic conditions.


METHODS
ECV-304 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 degrees Celsius; in the presence of 5% CO2. Liposome-ODN complexes were prepared just before use and added to the cells with a liposome-ODN charge ratio of 2:1. ECV-304 cell monolayers were transfected with liposome-ODN complexes containing 0.50, 0.75, 1.00 and 1.25 micromol/L ODN respectively in Euro-Collins solution (ECs) at 4 degrees celsius; and then stored for 2, 4, 6 and 8 hours respectively under anoxic condition. The ODN without liposome was transfected into ECV-304 cells under identical conditions as the control. The distribution of fluorescein isothiocyanate (FITC)-labeled ODN in ECV-304 cells was observed by fluorescence microscope, and the transfection efficiency and mean fluorescence intensity (MFI) were evaluated by flow cytometry.


RESULTS
MFI was enhanced as the storage time extended and ODN concentration increased, reaching the peak level at 6 h (P<0.05). After a 6-hour storage, most of the ODN was found to locate in the cell nuclei, and the transfection efficiency did not vary significantly between the groups. Compared with the control group, however, the differences in transfection efficiency and MFI were significant.


CONCLUSIONS
ODN can be highly efficiently transfected into ECV-304 cells by in vivo liposome in ECs under hypothermic and anoxic conditions, which provides an experimental basis for further study of the donor organ preservation at the level of genetic regulation.
Feng
Gui-wen
GW
Department of Kidney Transplantation, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
Yu
Li-xin
LX
Zhang
Hong-tao
HT
Zhao
Xian-guo
XG
D015687:Cell Hypoxia; D002460:Cell Line; D003080:Cold Temperature; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D009841:Oligonucleotides / Q000008:administration & dosage / Q000494:pharmacology*; D014162:Transfection / Q000379:methods*; D014471:Umbilical Veins / Q000378:metabolism
D008081:Liposomes; D009841:Oligonucleotides