Vitro Mechanism Exploration Of Hypoxia Inducing Factor 1-α On Angiogenesis Of Cerebrovascular

Vascular injury after traumatic brain injury as the key primary event could cause secondary injury,leading to dysneuria and death,which is the key link in the process of TBI repairment.Successful revascularization provides the key nerve vascular microenvironment for regeneration and reshape of neurons.Therefore,efficient strategies of promoting angiogenesis of TBI treatment gain more and more human attention.Hypoxia-inducible factor-1α is a transcriptional regulation factor,which could regulate the transcription of target genes,such as VEGF,EPO and i NOS,which makes it the regulation center of vascular response system in low oxygen conditions.Related studies have confirmed that HIF-1α could induce the formation of new blood vessels in rats ischemic myocardial injury and the brain injury model,meanwhile the mechanism of HIF-1α in angiogenesis is unknown.This study mainly explores the function and mechanism of HIF-1α in angiogenesis in vitro by b END.3 cell lines.Part 1 Experimental study of transfecting HIF-1 α into b END.3Objective:Transfect HIF-1 α into b END.3 in order to provide research methods for further research of HIF-1α role and its potential mechanism in cerebrovascular angiogenesis.Methods: Groups: control group(CON),GFP transfection group(NEGAT),HIF-1 α transfection group.Immunochemical method were used to validate b END.3.Transfect HIF-1 α to b END.3 by adenovirus.Results: Green fluorescence appeared at 24 h after transfection.It enhanced most at 2-3d after transfection,and it decreased to normal level at 7d.Transfection efficiency was about 85%.Western blot test showed no obvious expression of HIF-1α protein was detected in control group and GFP group.HIF-1α protein expression began to increase at 6h after transfection,and it reached the peak during 2-3d and began to decline after 3d and ultimately recovered to normal level at 7d.Conclusion: High efficiency of transfection conforms that adenovirus is a reliable transfection.Which could provide the research tools for further research of HIF-1α’s role and its possible mechanism in cerebrovascular angiogenesis.Part 2 Growth of b END.3 and expression of angiogenesis related factors after transfectionObjective: To observe the growth of b END.3 and detect expression of HIF-1 alpha and its downstream angiogenesis related factors such as vascular endothelial growth factor(VEGF),induced the expression of nitric oxide synthase(i NOS)after HIF-1α transfection.To explore potential mechanisms of angiogenesis promotion by HIF-1α.Methods: MTT was used to observe the proliferation of b END.3 cells.Q-PCR and Western blot were utilized to detect the expression of HIF-1α,i NOS and VEGF.Results: b END.3 began to increase in control group,GFP group and HIF-1α transfection group after incubation,with remarkable significance(P < 0.05).Increase rate of HIF-1α transfection group was higher than the other two groups and it came to the peak at 2-3d(P< 0.05).Migration ability of b END.3 was stronger in HIF-1α transfection group than the other two groups and it came to the peak at 2-3d(P< 0.05).Compared with the other two groups,Q-PCR and western blot test of HIF-1α transfection group showed expression of HIF-1α,i NOS and VEGF protein began to increase at 6h after transfection,and they reached the peak during 2-3d and began to decline after 3d and finally recovered to normal level at 7d.There was no significant difference between control group and GFP transfection group(P >0.05).Conclusion: HIF-1α transfection induces expression of its downstream gene such as i NOS and VEGF,which could promote angiogenesis by promoting b END.3 proliferation and migration.