Objective: To culture human retinal capillary endothelium cells (HRCECs) in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs.
Methods: Retinas were digested by 2.5% trypsin and 0.1% collagenase I in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium (HE-SFM BGM) with 10% fetal bovine serum, insulin-transferrin-selenium (ITS) and endothelial cell growth factor (ECGF). The cultured cells were identified by anti-factor VIII related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscopy, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs.
Results: Cultured HRCECs attached in the bottom of dishes in 48 h - 72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor VIII. EGPF positive cells were seen under laser confocal scanning microscopy after 48 h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085 ± 0.021 in normal group and 0.166 ± 0.024 in hypoxia group (t = 3.938, P < 0.05). In normal oxygen condition, the numeric value OA was 0.171 ± 0.011 in normal control group, 0.178 ± 0.016 in rAAV2-EGFP treated group, and 0.169 ± 0.017 in rAAV2-PEDF treated group (F = 0.01, P > 0.05). In hypoxia condition, the numeric value OA was 0.166 ± 0.013 in CoCl(2) treated group, 0.155 ± 0.012 in CoCl(2) + rAAV2-EGFP treated group, and 0.116 ± 0.015 in CoCl(2) + rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2.3% in normal control group, and 3.3% in rAAV2-EGFP treated group, and 1.7% in rAAV2-PEDF treated group. In hypoxia condition, the ratio of apoptosis was 3.6% in CoCl(2) treated group, 6.7% in CoCl(2) + rAAV2-EGFP treated group, and 36.4% in CoCl(2) + rAAV2-PEDF treated group.
Conclusions: PEDF gene can stably express in HRCECs after rAAV2-PEDF transfection and can obviously inhibit proliferation of HRCECs in hypoxia.