RT-qPCR quantification of mRNAs specific of Pigment Epithelium-Derived Factor (PEDF) and Vascular Endothelial Growth Factor (VEGF) in Rat eyes injected in the subretinal space with Primary Cells transfected with hPEDF.

摘要

Purpose : The aim of this study was to quantify human and rat PEDF, rat VEGF mRNAs in a model of laser-induced choroidal neovascularization (CNV) transfected with SB100X transposase with PEDF transposon encoding pFAR4 plasmids using real-time quantitative RT-PCR (RT-qPCR). Methods : Retinal pigment epithelia cells (RPEs) and iris pigment epithelia cells (IPEs) were transfected with pFAR4-pigment epithelium-derived factor (PEDF) plasmid using the non-viral Sleeping Beauty (SB100X) transposon system (pFAR4-ITRs CMV PEDF BGH-pFAR4-CMV SB100x SV40) in the subretinal space of Brown Norway rats. The treatment groups were as follows: not injected, injected with PBS, injected with the Venus plasmid (GFP), injected with the human PEDF plasmid or injected with the human PEDF plasmid + the SB plasmid. Total RNA was extracted from the injected eyes and purified for reverse transcription. Human PEDF, rat VEGF and rat PEDF mRNAs were quantified in eyes of rats using real-time quantitative RTqPCR. Relative quantity from each RNA was calculated for human and rat PEDF or rVEGF Ct normalized to Ct of RPL30 and have been determined with the Relative Quantification software from Applied Biosystems v1.2 after analysis of each qPCR plate with the SDS v2.3 software. Results : There were no significant differences in the mRNA expression in rat VEGF and PEDF for any group analyzed. Human PEDF was detected only in eyes injected with the hPEDF plasmid, with or without SB. Conclusions : Human PEDF was detected in rat eyes injected with the hPEDF plasmid demonstrating the efficacy of this SB system to release the PEDF. This system could be useful as an antiangiogenic therapy for retinal diseases. This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.