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Abstract
Objective: To investigate the efficiency of the transfection of PEGFP-IL-1ra plasmid via cation polymer mediation (poly-ethylenimine, PEI) by injection into the corneal stroma.
Methods: Plasmid PEGFP-hIL-1ra recombinants were constructed and transferred into corneal endothelial cells via cation polymer mediation. Plasmid PEGFP-hIL-1ra recombinants and/or PEI-in-vivo were injected into the corneal stroma of Wistar rats. Corneas were harvested at different time points (days 3, 6, 14 and 21) after injection. The expression of IL-1ra after transfection was studied by fluorescence microscopy, transmission electron microscopy, real time polymerase chain reaction (RT-PCR) and immunohistochemistry.
Results: Plasmid PEGFP-hIL-1ra recombinants were constructed successfully. After injection of pEGFP-hIL-1ra plasmid into the cornea, IL-1ra mRNA expression was detected in the corneal stroma and reached a peak on day 6. IL-1ra-GFP granules could be observed in every layer of the cornea in the PEGFP-hIL-1ra recombinant group by transmission electron microscopy, but not in the negative control (PEI-in-vivo) group. P63 immunocytochemical staining in the corneal epithelium was positive in both groups. There was no impairment in the ultrastructure of cells in both groups.
Conclusions: By direct injection of PEGFP-hIL-1ra into the corneal stroma and mediation by the cation polymer, the IL-1ra gene could be transferred and expressed in corneal tissue efficiently. This may be a novel technique for gene transfection to the cornea in situ.
Declaration of interest: This study was funded by the Natural Science Foundation of China (30801263) and the Natural Science Foundation of Guangdong Province (9451008901002232, 7001678).