[Transfection of brain-derived neurotrophic factor gene by recombinant adeno-associated virus vector in retinal ganglion cells in vitro].
OBJECTIVE
To determine whether rat retinal ganglion cells (RGCs) could be infected by rAAV-BDNF in vitro and to evaluate the influence of rAAV-BDNF transfection on the survival and apoptosis of rat RGCs.


METHODS
It was a experimental study. (1) RGCs were isolated from neonatal Sprague-Dawley rats (postnatal within 24 h). (2) Two days after the cultivation, the RGCs were transfected with rAAV-BDNF at a dosage of MOI = 10(5) and then incubated for 7 days. Total RNA were extracted from rAAV-BDNF transfected cells using Trizol reagent. The gene expression of BDNF gene in RGCs was analyzed by reverse transcription polymerase-chain reaction (RT-PCR). (3) Supernatant of the rAAV-BDNF transfected cells was collected at 7 days and 14 days after transfection. The protein expression of BDNF in the cell supernatant was examined with ELISA assay. (4) The survival and apoptosis of rAAV-BDNF transfected cells, untransfected cells and the cells with addition of BDNF in culture medium were evaluated by MTT colorimetric assay and flow cytometry with Annexin V-FITC staining, respectively.


RESULTS
(1) RT-PCR analysis showed that mRNA expression of BDNF gene could be detected in transfected cells but not in untransfected cells. (2)The concentrations of BDNF protein in the conditioned medium of the rAAV-BDNF transfected cells were (616.1 +/- 40.0) ng/L and (1075.1 +/- 48.7) ng/L 7 days and 14 days after the transfection, respectively. (3)MTT colorimetric assay showed that the OD values of rAAV-BDNF transfected cells and untransfected cells were similar at the time of 3 and 6 days after transfection( t = 1.084 and 1.582, P = 0.284 and 0.120). The OD value of transfected cells was higher than that of untransfected cells 9 days after the transfection (t = 4.854, P = 0.000). (4) The apoptosis rate in rAAV-BDNF transfected cells and the cells with BDNF exposure was lower than that of the untransfected cells (P = 0.015, 0.017).


CONCLUSIONS
Rat RGCs are able to be transfected by rAAV-BDNF in vitro. The transfected cells can express BDNF gene at the level of both mRNA and protein. Apoptosis rate is low in the transfected cells. This study indicates that rAAV-BDNF transfection can be used for the potential gene therapy in glaucoma neuroprotection.
Department of Ophthalmology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China.
Hai-yan
HY
Li
Jia-liang
JL
Zhao
Hua
H
Zhang
D000818:Animals; D017209:Apoptosis; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics*; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D014162:Transfection
D019208:Brain-Derived Neurotrophic Factor