β endothelial cell growth factor

Human eyes were obtained from the Eye Bank of Zhongshan Ophthalmic Center of Sun Yat-sen University within 24 h postmortem. All the donors of the eyes were healthy accident victims. The acquisition of all human materials complied with the ethical principles of the World Medical Association (Declaration of Helsinki) for medical research. The cell culture procedures were carried out as previously described [12 (link)]. Briefly, retinal tissues were removed by dissection and digested with 2% trypsogen and 0.1% collagenase I (Sigma Chemical Co, St. Louis, MO) for 20 min at 37 °C and then subjected to centrifugation (1,000 ×g for 10 min). The pellets were cultured in a 21.5-mm² culture dish coated with 5 mg/ml of fibronectin (Gibco, Grand Island, NY) for 1 h in human endothelial serum-free medium (Gibco), supplemented with 10% fetal bovine serum and 5 ng/ml β-endothelial cell growth factor (Sigma), in a humidified atmosphere of 5% CO₂ at 37 °C. The expression of the endothelial marker of cultured HRECs, anti-VIII factor antibody (Biosynthesis Biotechnology Co, Beijing, China) was determined by immunofluorescent staining. Only cells at passages 3 to 5 were used for the experiments. The HRECs were incubated with normal medium (control group: 5 mmol/l of glucose) or high glucose (high-glucose group: 30 mmol/l of glucose) for 72 h.