| BackgroundIschemic cerebrovascular disease is a commom and frequent disease with high mortality and maimed rate in clinic. It has been brought serious mental and economic burden to society, family and person. It has become the focus of research in the medical field that effective approaches to treat ischemic cerebrovascular disease are taked to reduce the patient's degree of disability and to improve their quality of life. Therefore, a novel treatment to ischemic cerebrovascular disease via inducing neoangiogenesis in ischemia area followed by better microcirculation in ischemia area and more blood supply of the affected brain tissue contributes to protect neurons in ischemic penumbra. The role of neuroimflamation in hypoxia-reoxygenation injury is complicated. Some inflammatory factors such as interleukin-1β (IL-1(3) exert opposing actions of neurological impairment and neuro-protection. So it is an important subject how to reduce its neurological impairment and to increase its neuro-protection.ObjectiveTo investigate the effect of IL-1(3on neoangiogenesis in a cellular model of rat brain microvascular endothelial cell (BMEC) induced by hypoxia-reoxygenation injury.Methods(1) Isolated and purified BMEC was identified by immunofluorescence of Ⅷfactor related antigen and its cell viability was evaluated with MTT chromatometry and cell growth curve.(2) The effect of different concentrations of IL-1β on BMEC viability in normal culture state was analysized by MTT assey.(3) The purified BMEC was cultured in anaerobic incubator for4hours and then reoxygenated for certain hours so as to establish hypoxia-reoxygenation models.(4) To study the effect of IL-1β induced by hypoxia-reoxygenation injury on expression of VEGF and MMP-9, a monoclonal neutralizing antibody against IL-1β was used in this experiment.(5) In order to investigate the effect of exogenous IL-1(3on cultured BMEC treated with hypoxia-reoxygenation injury, there were3groups in experiment: control group, hypoxia-reoxygenation group and hypoxia-reoxygenation group with IL-1β. Supernatant concentration of VEGF and MMP-9proteins was measured by enzyme-linked immunosorbent assay and cellular level of mRNA expression of VEGF and MMP-9was determined by real-time fluorescent quantitative PCR (QRT-PCR).(6) A three-dimensional culture system was established to observe the role of exogenous IL-1(3on tubule-like structure in tube formation assay of BMEC treated with hypoxia-reoxygenation injury.Results(1) The purity and yield of BMEC was satisfied with the following experiment. The liability of BMEC reached the top of the cell growth curve120hours after the beginning of cultivation.(2)A MTT assay showed lng/ml of IL-1(3reduced BMEC viability12hours after administration.(3) Supernatant VEGF and MMP-9concentrations were significantly induced by0.1ng/ml IL-1β in BMEC treated with hypoxia for4hours followed by reoxygenation for72hours.(4)0.1mg/ml IL-1β elevated VEGF mRNA levels by1.8-folds and MMP-9mRNA levels by2.0-folds in BMEC cells treated by hypoxia for4hours followed by reoxygenation for48hours (5) After addition of monoclonal neutralizing antibody against IL-1β, no significant change was found in supernatant VEGF or MMP-9levels in BMEC cultures treated with hypoxia for4hours followed by reoxygenation for72hours.(6) Tubule-like structures were markedly induced by0.1ng/ml IL-1β in BMEC cells treated with hypoxia for4hours followed by reoxygenation for72hoursConclusionsIn summary, this study revealed exogenous IL-1β can upregulate VEGF and MMP-9expression and improve formation of tubule-like structures in BMEC models treated with hypoxia-reoxygenation injury. |
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