Transfection of human umbilical vein endothelial cell line ECV-304 with liposome-oligonucleotide complexes under hypothermic and anoxic conditions

Abstract

Abstract: Objective To investigate the transfection efficiency of nuclear factor (NF)-κB 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 ℃ 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 μmol/L ODN respectively in Euro-Collins solution (ECs) at 4℃ 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. Conclusion 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.

CLC Number:

FENG Gui-wen¹, YU Li-xin¹, ZHANG Hong-tao², ZHAO Xian-guo². Transfection of human umbilical vein endothelial cell line ECV-304 with liposome-oligonucleotide complexes under hypothermic and anoxic conditions[J]. Journal of Southern Medical University, 2004, 24(05): 497-500.

References

[7] Ingrid HCV, Roland G, Hermann JG, et al. NF-κB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts [J].FASEB J, 2000, 14(7): 815-22
[1] Harvery PR, Iu S, McKeown CM, et al. Adenine nucleotide tissue concentrations and liver allograft viability after cold preservation and warm ischemia[J]. Transplantation, 1988,45(6): 1016-20.
[2] Gibbons GH, Dzau VJ. Molecular therapies for vascular disease[J].Science, 1996, 272(5262): 689-93.
[3] Barnes PJ, Karin M. Nuclear factor-κB -a pivotal transcription factor in chronic inflammatory disease [J]. New Eng J Med, 1997, 336:1066-71.
[4] Collins T. Endothelial nuclear factor-kappa B and the initiation of the atherosclerotic lesion[J]. Lab Invest, 1993, 68(4): 499-508.
[5] Cines DB, Pollak ES, Buck CA, et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders [J]. Blood, 1998,91(363): 3527-61.
[6] Tomita N, Morishita R, Higaki J, et al. A novel strategy for gene therapy and gene regulation analysis using transcription factor decoy oligonucleotides[J]. Exp Nephrol, 1997, 5(4): 429-34. .
[8] Azuma H, Tomita N, Kaneda H, et al. Transfection ofNF κ B-decoy oligodeoxynucleotides using efficient ultrasound-mediated gene transfer into donor kidneys prolonged survival of rat renal allografts [J]. Gene Ther, 2003, 10(4): 415-25.
[9] Tomita N, Morishita R, Tomita S, et al. Transcription factor decoy for NF kappa B inhibits TNF-alpha-induced cytokine and adhesion molecule expression in vivo[J]. Gene Ther, 2000, 7(13): 1326-32.
[10] 郑刚,张积仁,刘发全,等.脂质体介导al,4-GalT反义寡核苷酸对人脑胶质瘤细胞系SWO-38的作用[J].第一军医大学学报,2002,22(4): 299-302.Zheng G, Zhang JR, Liu FQ, et al. Effects of lipofectin-mediated a1 ,4Gal T antisense oligonucleotide on human glioma cell line SWO-38 [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2002,22(4): 299-302.
[11] 陈永鹏,章廉,胡俊,等.脂质体的乳糖化修饰及其肝脏靶向性研究[J].第一军医大学学报,2001,21(3):177-9.Chen YP, Zhang L, Hu J, et al. Study of lactosaminated liposome and its hepatotropic targeting in rats[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2001, 21(3): 177-9.
[12] 胡俊,章廉,冯筱榕,等.一种新型脂质体的制作方法[J].第一军医大学学报,2001,21(2):138-9Hu J, Zhang L, Feng XR, et al. A method for producing a new type ofliposome [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2001, 21(2): 138-9.
[13] Immai E. Gene therapy approach in renal disease in the 21 st century [J]. Nephrol Dial Transplant, 2001,16(Suppl 5): 26-34.
[14] Liu F, Qi H, Huang L, et al. Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration [J]. Gene Ther, 1997, 4(5): 517-23.
[15] Imai E, Isaka Y. Gene therapy for kidney disease[J]. Expert Opin Invest Drugs, 2000, 9(10): 1029-39.
[16] Morishita R, Sugimoto T, Aoki M, et al. In vivo transfection of cis element "decoy" against nuclear factor-kappa B binding site prevent myocardial infarction [J]. Nat Med, 1997, 3(8): 894-9.
[17] Zabner J, Fasbender AJ, Moninger T, et al. Cellular and molecular barriers to gene transfer by a cationic lipid[J]. J Biol Chem, 1995,270(54): 18997-9007.
[18] Noguchi A, Furuno T, Kawaura C, et al. Membrane fusion plays an important role in gene transfection mediated by cationic liposomes [J]. FEBS Lett, 1999, 433(2): 169-73.
[19] 胡英,金一,王华,等.阳离子膜融合脂质体介导反义寡核苷酸在Hela细胞中的转染试验研究[J].药学学报,2002,37(11):892-6.Hu Y, Jin Y, Wang H, et al. Study on the transfection efficiency of antisense oligodeoxynucleotide against telomerase by cationic fusogenic liposome[J]. Acta Pharm Sin, 2002, 37(11): 892-6.
[20] Nakanishi M, Mizuguchia H, Ashihara K, et al. Gene transfer vectors based on Sendai virus[J]. J Control Rel, 1998, 54(1): 61-8.