科研之友
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摘要
Background Hydrodynamic gene delivery has proved an efficient strategy for nonviral gene therapy in the murine liver but it has been less efficient in pigs. The reason for such inefficiency remains unclear. The present study used a surgical strategy to seal the whole pig liver in vivo. Methods A solution of enhanced green fluorescent protein (eGFP) DNA was injected under two different venous injection conditions (anterograde and retrograde), employing flow rates of 10 and 20 ml/s in each case, with the aim of identifying the best gene transfer conditions. The gene delivery and information decoding steps were evaluated by measuring the eGFP DNA, mRNA and protein copy number 24 h after transfection. In addition, gold nanoparticles (diameters of 4 and 15 nm) were retrogradely injected (10 ml/s) to observe, by electron microscopy, the ability of the particle to access the hepatocyte. Results The gene delivery level was higher with anterograde injection, whereas the efficacy of gene expression was better with retrograde injection, suggesting differences in the decoding processes. Thus, retrograde injection mediates gene transcription (mRNA copy/cell) equivalent to that of intermediate expression proteins but the mRNA translation was lower than that of rare proteins. Electron microscopy showed that nanoparticles within the hepatocyte were almost exclusively 4 nm in diameter. Conclusions The results suggest that the low activity of mRNA translation limits the final efficacy of the gene transfer procedure. On the other hand, the gold nanoparticles study suggests that elongated DNA conformation could offer advantages in that the access of 15-nm particles is very limited.
- 出版日期2014-8
