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Background and purpose of the present that in acute ischemic renal failure (iARF) pathogenesis of many inflammatory cytokines such as: endothelin -1 (ET-1), inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein -1 (MCP-1), intercellular adhesion molecule -1 (ICAM-1) and vascular cell adhesion molecule -1 (VCAM-1) and other involved in the occurrence of renal damage. These genes contain inflammatory mediators (B locus, are generated by the transcription factor NF-κB regulation. Vivo transfection of the NF-κB binding site oligonucleotides (NF-κB decoy ODN) technology specifically inhibit its activity, this strategy has been applied in the treatment of certain diseases, studies, and shows a broad prospect, but what on cultured renal tubular epithelial cells how, there is no report of this study aims to establish an efficient and stable protamine - liposomes (protamine liposome, PL) transfection technique, based on the observation of NF-κB decoy ODN on renal tubular epithelial cells NF-κB activity and the expression of inflammatory molecules downstream affected. Methods NRK-52E renal tubular epithelial cells by TNF (cultured in vitro stimulation experiments in three parts: the establishment of protamine - lipofection NF-κB decoy ODN technology (protamine liposome, PL law): The NF-κB decoy ODN with protamine, liposomes mixed transfected into TNF (stimulation of cultured rat serum containing complete renal tubular epithelial cells, transfection efficiency was measured. using gel electrophoresis retardation assay (EMSA ) Determination of transfected NF-κB decoy ODN after cell NF-κB activity was detected by RT-PCR transfected NF-κB decoy ODN after cells ET-1, iNOS, ICAM-1, VCAM-1 and MCP -1 mRNA expression levels were ODN cellular uptake rate and distribution: The PL France transfected NRK-52E cells, 2 h after the strong fluorescence seen in the nucleus particle distribution, while the bare transfected ODN, the nucleus has been no clear fluorescence distribution by FCM analysis, the PL transfected 2 h after transfection rate was 93.20%, while the bare transfection rate was 39.03% for the. by TNF (stimulation for 30 min, NRK-52E cells, NF- κB activity began to increase, and with the stimulus lt; WP = 5 gt; increasing the extension of time, to stimulate the activity reached a peak after 6 h, 12 h after the stimulus began to fall in the transfection of NF-κB decoy ODN After 6 h, the cells of the NF-κB activity was inhibited. by TNF-(stimulated cultured NRK-52E cells, ET-1, iNOS, ICAM-1,, VCAM-1 and MCP-1 mRNA expression was significantly increased , with and without TNF-(stimulated cultured group, the difference was significant, P (0.01; Transfected NF-κB decoy ODN, these molecules mRNA expression was significantly inhibited, and TNF-(stimulated cultured group, P (0.01 Conclusion PL method is an efficient cationic transfection techniques, to overcome the majority of cationic liposomes can not be used under serum-containing conditions limit transfected can successfully NF-κB decoy ODN binding sites transfected into cultured renal tubular epithelial cells, this method further research, will have broad application prospects in cultured NRK-52E renal tubular epithelial cell lines, NF-κB decoy ODN can specifically suppressed by TNFα activated NF-κB, and no mismatch scrambled ODN this effect, the NF-κB decoy ODN strategies can study the role of NF-κB-specific means of the activation of NF-κB can be increased ET- 1, iNOS, MCP-1, ICAM-1 and VCAM-1 expression of inflammatory factors such as NF-κB by NF-κB decoy ODN inhibited, the expression of these factors is accordingly suppressed. Accordingly, NF-κB decoy ODN strategy can prevent NF-κB activity, thereby inhibiting a variety of regulated expression of inflammatory cytokines. further study NF-κB decoy ODN on renal tubular epithelial cell injury, including ischemia as this policy is expected to ARF in the treatment of a variety of diseases, including the new means.
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