HIF-1¦Á Impact Of Migration And Differentiation Of Neural Stem Cells After Cerebral Infarction, And Experimental Study Of The Mechanism

Objective: To investigate the effect of hypoxia-inducible factor-1αgene on the proliferation and differentiation of neural stem cells and the angiogenesis after focal cerebral ischemia in rats, and to explore the mechanism of the effect.Methods: We utilized a transient middle cerebral artery occlusion (tMCAO) model. 1 day later, the rats with NSS scores from 7 to 12 were randomly divided into NS group, Ad group and Ad-HIF-1αgroup. NS, Ad and Ad-HIF-lαwere injected into the ischemic ventricle respectively, BrdU was used to lable the proliferation and differentiation of NSCs. (1)NSS scores were evaluated and compared at days 7, 14, 21 and 28. (2) HIF-1αand VEGF mRNA were detected at hours 6, 12 and days 1, 2, 3 and 7 after injection. The expression of HIF-1αand VEGF was evaluated with immunohistochemistry at days 2 and 7. The expression of EPO in peri-ischemic tissue was observed using immnostaining. (3) Both the number of BrdU positive cells in subventricular zone at different time points after reperfusion and the number of BrdU/NF200, BrdU/GFAP double labeled cells in cortex were calculated using immumofluorescence method. (4) The expression of factorⅧwas detected using immunostaining at days 28.Results(:1)NSS scores of Ad-HIF-1αgroup were superior to those of NS group and Ad group at days 7,14,21,and 28(P<0.05). (2)The expression of HIF-1α, VEGF and EPO was higher in Ad-HIF-lαgroup than those in the other two groups; (3) The number of BrdU positive cells in Ad-HIF-lαgroup was increased obviously in Ad-HIF-lαgroup. The cellular rebirth and differentiation demonstrated that it was significant difference(P<0.05) to compare BrdU/NF200(47.74%±13.52%) and BrdU/GFAP(67.83%±20.75%) of Ad-HIF-lαgroup with those of the other two groups at the 28 d.(4) It was significantly different to compare the number of factorⅧpositive cells of Ad-HIF-1αgroup with those of Ad group (p<0.01).Conclusion: These results indicate that HIF-1αgene can increase proliferation and differentiation of endogenetic NSCs and promote angiogenesis, and thus improve the recovery of neurofunction. Objective: To observe the expression of Hypoxia-inducible factor-1αin neural stem cells infected by adenovirus carrying with HIF-1αgene and the effects on bionomics of the infected cells.Methods: We cultured NSCs of the cerebral cortex in fetal rats.Then the NSCs were transferred by recombinant adenovirus with with GFP and HIF-1αgene. After that, the morph of NSCs was observed under phase contrast microscope, and the expression of GFP both before and after the differentiation of NSCs was observed under fluorescent microscope. The expression of HIF-1αin NSCs was detected by western blot. The special protein such as nestin, neurofilament(NF), glial fibriliary acidic protein(GFAP) were detected with immunofluorenscence. We Used clampex to observe sodium current and the anoxic tolerance of neuron differentiated from NSCs.Results: (1) After transfection the exogenous HIF-1αgene and GFP can be expressed continuously and steadily in NSCs. (2) The morphological feature of the transferred NSCs was similar to that of the untransferred NSCs. The transfection promoted the proliferation of NSCs. (3) The transferred NSCs were confirmed to have the latent ability of multi-directional differentiation identified by NF and GFAP immunostaining. (4) It was significantly different to compare the sodium current of neuron differentiated from HIF-1αinfected NSCs(1053.7士127.4 pA) with that of neuron differentiated from Ad infected NSCs(637.2士82.5 pA) and with that of neuron differentiated from uninfected NSCs(597.3士73.5 pA)(P<0.01). The time of anoxic tolerance was prolonged obviously.Conclusion: (1) The adenovirus which carring with HIF-1αand GFP can transfer NSCs efficiently in vivo. NSCs can express HIF-1αand GFP steadily after infected. (2) The expression of HIF-1αcan promote the proliferation of the infected cells and can improve sodium current and the ability of anoxic tolerance of neuron differentiated from NSCs. Objective: To observe the effects of transplanting HIF-1αgene modified NSCs on the cell survival and migration and angiogenesis after focal cerebral ischemia, and to explore the mechanism.Methods: We utilized a transient middle cerebral artery occlusion (tMCAO) model. 1 day later, the rats with NSS scores from 7 to 12 were randomly divided into NS group, NSCs-Ad group and NSCs-HIF group. NS, NSCs-Ad and NSCs-HIF were injected into the ischemic ventricle respectively.All NSCs have labeled by BrdU for 3 days before transplantation. NSS scores were compared at days 7, 14, 21 and 28. HIF-1αexpressed by NSCs-HIF was detected using double labeled method. The numbers of transplanted NSCs in two-side cortex was caculated at weeks 5 after injection. FactorⅧimmunostaining was performed at weeks 5. The expression of slit2 was evaluated at different time points.Results: (1) NSS scores of NSCs-HIF group were superior to that of NS group and NSCs-Ad group at days 7,14,21,and 28(P<0.05). (2) NSC-HIF expressed HIF-1αin vitro.(3) The number of transplanted NSCs in ischemic cortex was increased obviously in NSCs-HIF group(P<0.01); (4) The number of factorⅧpositive cells in NSC-HIF group was significantly different from that in NS group and from that in NSCs-Ad group (p<0.01). (5) Focal cerebral ischemia could induce the express of slit2 in the ischemic cortex. NSCs-HIF increased the Slit2 expression at different time points.Conclusion: HIF-1αgene has therapic effect on focal cerebral ischemia through promoting the survival and migration of NSCs and increasing the angiogenesis.