Effect of rAAV2-PEDF on migration and apoptosis of human retinal capillary endothelial cells

AIM: To construct a rAAV2-pigment epithelium-derived factor (PEDF) vector and to explore the effect of rAAV2-PEDF on the migration and apoptosis of human retinal capillary endothelial cells (HRCECs). METHODS: PEDF gene was cloned into an adeno-associated virus vector pSNAV. The recombinant pSNAV-PEDF was transfected into BHK cells. The G418 resistant cells were selected and infected with recombinant HSV which packaged the recombinant pSNAV-PEDF. The high-titer rAAV2-PEDF was obtained by purification. After transfected the rAAV2-PEDF into HRCECs, the protein expression of PEDF were detected by Western blotting, the migration of HRCECs was assessed by a boyden chamber, and the apoptosis of HRCECs was analyzed by flow cytometry. RESULTS: The evaluation results of PCR, enzyme digestion, and DNA sequencing showed that the rAAV2-PEDF was constructed correctly. The GFP positive cells were observed under laser scanning confocal microscope 48 h after rAAV2-GFP transfection. The expression level of PEDF protein was higher in experimental group than that in control group. The number of migrated cells was 33.0±2.7 in normal control group, 35.0±3.6 in rAAV2-GFP treatment group, and 12.0±2.1 in rAAV2-PEDF treatment group (P<0.05). In normoxic condition, the apoptotic cells were 2.10%±0.53% in control group, 3.40%±0.62% in rAAV2-GFP group and 1.60%±0.47% in rAAV2-PEDF group (P>0.05). In hypoxic condition, the apoptotic cells were 4.00%±0.55% in CoCl₂ treatment group, 6.10%±0.71% in CoCl₂+rAAV2-GFP treated group and 40.00%±2.10% in CoCl₂+rAAV2-PEDF treatment group (P<0.05). CONCLUSION: rAAV2-PEDF is successfully constructed and PEDF gene is stably expressed in HRCECs after rAAV2-PEDF transfection. Over-expression of PEDF inhibits the migration of HRCECs and induces the cell apoptosis obviously in hypoxia.