Adeno-Associated Virus Mediated Gene Transfer Of Brain-Derived Neurotrophic Factor And Neurotrophin-4 To Rat Retinal Ganglion Cells In Vitro

Objective1. To establish a culture system for rat retinal ganglion cells (RGCs) in order to lay a foundation for the experimental research of RGCs in vitro.2. To evaluate the efficiency and safety of transduction of recombinant adeno-associated virus-mediated green fluorescence protein (rAAV-GFP) gene to rat retinal ganglion cells (RGCs) and so to decide the possibility of rAAV as a vector in the gene therapy for glaucoma neuroprotection.3. To determine whether rat retinal ganglion cells (RGCs) in vitro could be infected by rAAV-BDNF or rAAV-NT4 and to examine the expression of exogenous BDNF or NT4 gene in the level of transcription and translation.4. To evaluate the influence of rAAV-BDNF or rAAV-NT4 transfection on the survival and apoptosis of rat RGCs in vitro.Methods1. The retina of neonatal Sprague-Dawley rats (postnatal within 24h) were dissected into cell suspension with trypsin digestion. Culture plate were coated with Poly-L-lysine (PLL, 0.1mg/mL). After incubation for 24h, 5-bromo-2'-dexyuridine was added to the culture medium with a final concentration of 20μg/mL. Cells were cultured in an incubator with 5%CO2 under 37℃. The cells were observed under phase-contrast microscope every day.