RESUMO
Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and 19%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic fibroblast growth factor isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to 17% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Terapia Genética/métodos , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Transfecção/métodos , Animais , Sequência de Bases , Sobrevivência Celular/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microinjeções , Dados de Sequência Molecular , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/fisiologia , Epitélio Pigmentado Ocular/citologia , Plasmídeos/genética , Ratos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
We present a 26-year-old athlete with severe low back pain associated with a fracture of a lumbar pedicle and contralateral spondylolysis. The difficulties of diagnosis are discussed together with the subject of segmental pain referral. A technique of operative management is described involving instrumentation of the symptomatic level alone.
