Study On The Effect Of TNF-α On Angiogenesis In Ischemia-reperfusion

BackgroundIschemic cerebrovascular disease is a serious hazard to human health, it has become a problem for medical research and has caused problems to the world. Ischemia-reperfusion is organs and tissues can be reperfused after ischemia, furthermore, the organ function and damaged tissue can be restored. A new therapy appears to cure cerebral ischemia by promoting neovascularization to ameliorate the core necrotic region and peripheral ischemic penumbra ischemic.Inflammatory factor showe a greater complexity on neovascularization after ischemia-reperfusion.Tumor necrosis factor-a (TNF-α) show a dual role in angiogenesis. How to reduce alleviate the damage of brain tissues by TNF-α and enhance the protective effect on neurons, it is need to be solved urgently.ObjectiveTo determine the effect of TNF-α in an in vivo and vitro model of angiogenesisMethods(1) Rat cerebral microvessel endothelial cells (RCMECS) isolated and purified from SD rat. Immunomagnetic purification is performed using anti-PECAM-1(CD31) polyclonal antibody.(2) The method of MTT was used to examine activity of TNF-α to RCMECS in normal conditions (3) RCMECS treated TNF-α or neutralizing antibody against TNF-α in hypoxia(4hours)/reoxygenation (72hours) conditinon.S-upernatant concentration of VEGF and MMP-9proteins was measured by enzyme-linked immunosorbent assay.(4) Supernatant concentration of VEGF and MMP-2cellular level of mRNA expression of VEGF and MMP-2was determined by real-time fluorescent quantitative PCR (QRT-PCR), after reoxygenation for48hours.(5) The model of middle cerebral artery occlusion(MCAO) and reperfusion was established and the effects of TNF-α on angiogenesis in ischemic cortex of newborn was observed.Results (1) The purities were upto90%and yield of BMEC was satisfied (2) A MTT assay showed0.08ng/ml of TNF-α reduced BMEC viability24hours after administration.(P<0.05).(3)It can be observed that addition of TNF-α have significant difference compared H/R group,the opposite was true in the the addition of monoclonal neutralizing antibody against TNF-α group,after reoxygenation for72hours (P<0.05).(4) The results of this research find that0.08μg/ml TNF-a elevated VEGF mRNA levels by1.7-folds and MMP-2mRNA levels by1.9folds.(5) The model of middle cerebral artery occlusion(MCAO) and reperfusion was successfully established.(6) The greater number of newborn blood vessels TNF-a treated with RCMECS was observed, within7-21days after cerebral ischemia.(P<0.05). Conclusions:Exogenous TNF-α can upregulate VEGF and MMP-2expression in in vivo and vitro model, it can stimulate newborn blood vessel development after cerebral ischemia.