Hypoxia-inducible Factor-1a After Intracerebral Hemorrhage In Neural Stem Cell Proliferation, Migration, Differentiation, And Vascular Newborn Role Of Experimental Research

PARTⅠEFFECT oF ADENOVIRUS-MEDIATED HYPOXIA INDUCIBLE FACTOR-1ALPHA GENE ON TARGET GENES AFTER INTRACEREBRAL HEMORRHAGEObjective : To explore expression and significance of hypoxia inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF),erythropoietin ( EPO) in the perihematoma with intracerebral hemorrhage .Methods : Experimental ICH was induced by right intrastriatal administration of autologous whole blood in adult rats . 30 minutes after surgery, the rats were randomly divided into PBS group,Ad group and Ad-HIF-1 group . PBS,Ad and Ad-HIF-1a were injected into the hemorrhagic lateral ventricle respectively.HIF-la,VEGF and EPO mRNA were detected at hour 4,24,72 and 168 after injection.While the expression of HIF,VEGF and EPO protein were evaluated with immunohistochemistry . HIF-la and VEGF or HIF-la and EPO using double labeled method.Results:HIF-1a,VEGF and EPO mRNA levels in the perihematoma with ICH started to increase as early as 4 hours after ICH, peaked at 3 days, and then gradually decreased at 7 days. Immunohistochemistry also demonstrated that HIF-1a,VEGF and EPO protein immunoreactivity was increased in the the perihematoma with ICH. Ad-HIF-1a group significantly increased HIF-1 a,VEGF and EPO mRNA or protein levels in the ipsilateral basal ganglia compared with PBS group and Ad group(P<0.05), and there were no significant differences between PBS group and Ad group(P>0.05).Conclusion : This study demonstrates that perihematomal HIF-1a ,VEGF and EPO mRNA or protein is upregulated after ICH. Ad-HIF-1a gene can upregulate obviously the expression of HIF-1a ,VEGF and EPO mRNA or protein . Time-dependent relationship between HIF-1a and VEGF or HIF-1a and EPO. PARTⅡTHE EFFECT oF ADENOVIRUS-MEDIATED HYPOXIA INDUCIBLE FACTOR-1a GENE ON THE PROLIFERATION AND DlFFERENTIATION OF NSCs AND THE ANGIOGENESIS AFTER INTRACEREBRAL HEMORRHAGEObjective:To observe the effects of transplanting hypoxia inducible factor-1α(HIF-l a) gene on the endogenetic NSCs survival,migration,differentiation and angiogenesis after ICH,and to explore the mechanism of the effect.Methods : Experimental ICH was induced by right intrastriatal administration of autologous whole blood in adult rats. 30 minutes after surgery, the rats were randomly divided into Sham group,Ad group and Ad-HIF-1a group. PBS,Ad and Ad-HIF-1a were injected into the hemorrhagic lateral ventricle respectively. All endogenetic NSCs have been labeled by BrdU .The animals were evaluated for 4 weeks with NSS scores . The numbers of DCX,Brdu in two-side subventricular zone (SVZ)was caculated at week 1, 2, 3, 4 after injection.Results:(1) Ad-HIF-1a group showed better functional performance on NSS scores than Sham group and Ad group after days 7,14,21,28 (P<0.05). (2) DCX levels in the ipsilateral basal ganglia started to increase as early as 7 days after ICH, peaked at 14 days, and then gradually decreased at 1 month. Immunohistochemistry also demonstrated that DCX immunoreactivity was increased in the ipsilateral subventricular zone and basal ganglia at 2 weeks after ICH. Ad-HIF-1a group significantly increased DCX levels in the ipsilateral basal ganglia compared with Sham group and Ad group(P<0.01,P<0.05),and there were significant differences between Sham group and Ad group(P<0.01). Some DCX positive cells were BrdU-positive. (3) The number of BrdU positive cells in Ad-HIF-la group was increased obviously than that in the other two groups(P<0.01,p<0.05),and there were significant differences between Sham group and Ad group(P<0.01).Endogenetic NSCs migrated selectively to the perihematomal areas and differentiated into neurons and astrocytes , Ad-HIF-la group was increased obviously than that in the other two groups(p<0.05), there were significant differences between Sham group and Ad group(P<0.01). (4)Treatment with HIF-la gene increased angiogenesis ,as indicated by double staining of CD31 and Brdu. The number of CD31 positive cells in Ad-HIF-la group was significantly different from that in Sham group(p<0.0 1) and from that in Ad group(p<0.0 5), there were significant differences between Sham group and Ad group(P<0.05).Conclusion:These results indicate that HIF-1 a gene can increase proliferation,migration and differentiation of endogenetic NSCs and promote angiogenesis after ICH ,and thus improve the recovery of neurofunction. SECTION TWO ADENOVIRUS MEDIATED HIF-1αGENE MODIFYING NEURAL STEM CELLS OF MICE IN VITROPARTⅠISOLATION,CULTIVATION AND IDENTIFICATION OF NEURAL STEM CELLS FROM BRAIN CORTEX OF EMBRYONIC RAT IN VITROObjective : To isolate,cultureand and identify neural stem cells(NSCs)from brain cortex of embryonic rats in vitro . Methods:Neurons in cortex of embryonic rats were isolated and cultured in vitro.Differentiation conditions were optimal by exposing neurospheres to10% fetal bovine serum. The cells were fixed and stained by using Immunofluorescence to detect the specific antigen of neuron (NeuN), astrocyte(GFAP) , oligodendrocyte (MBP)and snaptophysin (GAP-43).Results:The cells derived from the brain cortex of embryonic rats expressed nestin antigen and have the potential to form clones and differentiate into neurons, astrocytes, oligodendrocytes and snaptophysin.Conclusion: The cells isolated from brain cortex Of embryonic rat exhibited the ability for self-renewal and multipotency,. supposing their character of neural stem cells.PARTⅡADENOVIRUS MEDIATED HIF-1αGENE MODIFYING NEURAL STEM CELLS OF MICE IN VITROObjective: To provide material foundation for the transplantion of neural stem cells (NSCs) modified by HIF-1αgene in the treatment of intracerebral hemorrhage of adult rats . we transfected NSCs using recombinant adenovirus with HIF-1αand GFP gene,and observed the expression of HIF-1αand GFP,and their effects on bionomics of the NSCs.Methods:NSCs were transfected with the recombinant adenovirus, and then the expression of HIF-1αand GFP in the NSCs was observed. NSCs were detected under phasecontrast microscope and fluorescent microscope. After successful transferring, the expressions of Nestin, BrdU, NeuN, GFAP, MBP in NSCs clone cell was examined with the immunofluorescent cytochemical method and immunohistochemical method, and the expression of the HIF-1αgene in the NSCs was detected with the immouncytochemical method and RT-PCR. Results:The exogenous HIF-1αgene could be expressed steadily in NSCs after transfection. The morphological features of the untransfected NSCs were similar to those of the transfected,and there were no significant differences between the transfected NSCs and the untransfected in the cytoactivity and differentiation (P>0.05). The transfected NSCs were confirmed to have the latent ability of multi-directional differentiation.Conclusion: HIF-1αand GFP could express stably in NSCs after transfected with the recombinant adenovirus and the bionomics is normal. The data demonstrate that the hypoxia HIF-1αtransduced NSCs might serve as a useful source of donor cells for the gene therapy of intracerebral hemorrhage. SECTION THREE THE EFFECT OF TRANSPLANTING HIF-la GENE MODIFIED NSCs ON CELL SURVIAL AND MIGRATION AND ANGIOGENESIS AFTER INTRACEREBRAL HEMORRHAGEObjective:To observe the effects of transplanting HIF-la gene modified NSCs on the cell survival,migration,differentiation and angiogenesis after intracerebral hemorrhage(ICH),and to explore the mechanism.Methods : Experimental ICH was induced by right intrastriatal administration of autologous whole blood in adult rats. One week after surgery, the rats were randomly divided into PBS group,Ad–NSCs group and HIF-NSCs group.PBS,Ad–NSCs and HIF-NSCs were injected into the hemorrhagic lateral ventricle respectively.All NSCs have been labeled by BrdU for 3 days before transplantation.The animals were evaluated for 8 weeks with NSS scores . Transplanted NSCs were detected by BrdU and HIF-l a,nestin,doublecortin,GFAP,NeuN and CD31 using double labeled method . The numbers of transplanted NSCs in two-side subventricular zone (SVZ)was caculated at week 8 after injection.Results:(1) HIF- NSCs group showed better functional performance on NSS scores after days 8,14,21,28, 35, 42, 49, 56 (P<0.05) compared with PBS group and Ad-NSCs group (P<0.05). (2) HIF-NSCs expressed HIF-la,nestin,doublecortin,GFAP and NeuN in vitro.(3) Transplanted HIF -NSCs migrated selectively to the perihematomal areas and differentiated into neurons (29%) and astrocytes ( 63%). (4 ) The number of transplanted NSCs in perihemotoma was increased obviously in HIF-NSCs group than Ad- NSCs group and PBS group(P<0.05) , while there were no significant differences between PBS group and Ad group(P>0.05). (5) Treatment with HIF-la-NSCs increased angiogenesis ,as indicated by double labeled staining of CD31 and Brdu. The number of CD31 positive cells in HIF-NSCs group was significantly different from that in PBS group and from that in Ad-NSCs group(p<0.0 1).Conclusion:Transplanted HIF-la-NSCs can enter the rat brain with ICH, survive, migrate, differentiate , increasing the angiogenesis and improve functional recovery. Transplantation of HIF-la-NSCs can be used to restore neurological deficits in experimental ICH.