ABSTRACT
Background Choroidal neovascularization(CNV)has been descrihed as a main reason of visual loss in a lot of ocular diseases.Researches showed that local hypoxia and retinal pigment epithelial(RPE) cells play an important role in the formation of CNV.A closely relationship of angiopoietin-2 (Ang-2) and angiogenesis has been proved.However,whether the expression of Aog-2 in hypoxic cultured human RPE cells is associated with the pathogenesis of CNV is still below understood.Objective This study was to investigate the effects of hypoxia on expression of Ang-2 in cultured human RPE cells in vitro,and discuss the possible effects of Ang-2 in the formation of CNV.Methods Human RPE cells were cultured and passaged,and 4-7 generation of cells were used in the experiment.The cells were incubated in cultural plate at the density of 5×107 cells/L.The culture medium containing 200 μmol/L CoCl2 was used to establish the hypoxia model of human RPE cells culturecd in vitro for 0.5,1,2,4,6,12and 24 hours,and the RPE cells cultured under normoxia were as controls.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of Ang-2 mRNA in cultured human RPE cells,and enzymelinked immunosorbnent assay(ELISA) was used to assay the content of Ang-2 protein in supernatant of cultured human RPE cells.Results The survival rate of human RPE cells was 90％ after resoscitation.The fourth generation of cells showed the fusiform with the less pigment in them.The Ang-2 mRNA/β-actin mRNA value in human RPE cells was significantly different among various groups(F=1086.30,P=0.00),The Ang-2 mRN A/β-actin mRNA value in hypoxia cultured for 0.5 hours group began to increase and peaked in hypoxia culture for 4-6 hours group,with the significant differences in comparison with normoxia control group(P＜0.05).The Ang-2 mRNA/β-actin mRNA value decreased to the baseline level at hypoxia for 24 hours.The ELISA analysis showed that the concentration of Ang-2 protein in supernatant of RPE cells showed significant difference among groups(F=1034.00,P=0.00).The concentration of Ang-2 protein increased at hypoxia culture for 0.5 hours and peaked at 6 hours,showing significant differences in comparison with the control group (P＜0.05).Conclusions Hypoxia could significantly up-regulate the expression of Ang-2 in human RPE cells cultured in vitro.Ang-2 expresses highly in the early stage of hypoxia,implying that Ang-2 participates in the formation of CNV.