ABSTRACT
Objective: To search for the optimizing parameters and distribution pattern of NF ?B decoy oligodeoxynucleotides in transfecting J774.1 cells mediated by lipofectin. Methods: With the change of ODNs/lipofectin ratio and transfection time, the uptake rate and mean fluorescence intensity of NF ?B decoy oligodeoxynucleotides in J774.1 cells were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of NF ?B decoy oligodeoxynucleotides was determined with fluorescence microscopy. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity. Results: The uptake of NF ?B decoy oligodeoxynucleotides into J774.1 cells was significantly improved by lipofectin. In 24 well culture plate, 1 ?g ODNs with 5?g lipofectin ( W/W =1∶5) resulted in the highest transfection efficiency and lower cytotoxicity. The NF ?B decoy oligodeoxynucleotides localized in the nucleus as well as in the cytoplasm following an incubation of 6 h with lipofectin. While NF ?B decoy oligodeoxynucleotides had faint fluorescences in cytoplasm in the absence of lipofectin. Conclusion: lipofectin can enhance the cellular uptake of NF ?B decoy oligodeoxynucleotides in J774.1 cells and alter intracellular distribution of NF ?B decoy oligodeoxynucleotides. Efficiency of transfection is the highest when the ratio of ODNs/lipofectin is 1∶5 for incubation of 6 h.
ABSTRACT
AIM: To study the effect of NF-?B “decoy”oligodeoxynucleotides on IL-10 expression in LPS-induced mouse macrophages. METHODS: Mouse macrophage cell line J774 1 cells were cultured with LPS and liposome-mediated oligodeoxynucleotides, and the level of IL-10 were measured in culture medium by enzyme linked immunosorbent assay. RNA was extracted from macrophages, and the mRNA expression of IL-10 were observed by RT-PCR. RESULTS: NF-?B “decoy”oligodeoxynucleotides did not inhibit the expression of IL-10 in LPS-induced macrophage, and did not reduce generation of IL-10. CONCLUSION: NF-?B decoy ODNs did not depress the mRNA expression of IL-10. NF-?B may not be the critical transcription factor in transduction mechanism of IL-10.