Transferring BFGF Gene To Cat Corneal Endothelial Cells In Vitro By SPIONs

Obiective:To observe the transfection efficiency of transferring bFGF gene encoding enhanced green fluorescent protein (EGFP) to cat corneal endothelial cells by SPIONs which modified by poly-L-lysine. In order to investigate the feasibility of the SPIONs as a new gene carrier for corneal endothelial cell in vitro.The study includes two parts:Part1:Study on the proliferation,differentiation and assay of cat corneal endothelial cells in vitro.Part2: Study on the pEGFP-C1-bFGF gene transfection to cat corneal endothelial cells and observation on its expression in cells.Method:Part1:Eyeballs were taken from the cat which the age is less than 3 months and their limbus were carefully cut to take transparency cornea in strict sterile working, then to culture the primary CECs by stripping descemet method. Cells in different cultured course were observed under the inverted microscope also under light microscope with trypan blue dying. NSE staining was used to identify the cultured CECs.Part2: poly-L-lysine(PLL)was linked on the surface of SPIONs by nanoparticle surface energy and electrostaticalIy binding,a novel complex nanomaterial SPIONs-PLL was prepared.Then the SPIONs-PLL conjugated with the enhanced green fuorescence protein DNA plasmid (pEGFP-C1-bFGF) was transfected into CECs.The efficiency was investigated in transfected cells by fluoromicroscopy examine and the expression of bFGF gene was revealed. ResultsPart1: Primary CECs were successfully cultured and continuously cultured into passage four in vitro.The primary and passage one cells continued amplification with regular polygon morphology grew better than passage two to four which showed the cells became senescent gradually. Percentages of living cells were 99.8% , 99.1% ,98.2%,94.3%,91.3% in the primary to passage four cells respectively. The cultured cells were stained with NSE and were observed positive expression in the cytoplasm.Part2: A novel positive charge nanomaterial SPIONs-PLL was sucssesfully prepared.The nanoparticles were spherical and uniform in size,highly monodispersed and stable because of charge rejection. pEGFP-C1-bFGF gene was successfully transferred into CECs.48 hours later specific green fluorescence was observed by fluorescence microscope in gene group. Specific green fluorescence was not be observed in control group.Cells of gene group and control group grew well.Western blot showed the band of bFGF emerged in gene group,and the expression of additional magnetic field group was stronger.Conclusions:1.Primary CECs were successfully cultured and passed with stripping descemet method in vitro.Reproductive activeity of passage cell gets worse progressively.The cultured cells were source of neural crest because of expressing positive in cytoplasm by NSE staining.2. The pEGFP-C1-bFGF gene is successfully transferred into CECs by SPIONs and the cells grow well.The transfection efficiency of additional magnetic field group was the highest ,so SPIONs could be used as a novel DNA carriers for gene transfection.