Effects Of Angiogenesis On The Proliferation And Differentiation Of Neural Stem Cells In The Subventricular Zone Of Neonatal Rat

[Object]Since the cerebrum of premature infant is in the developing stage.,how to use the feature that premature brain is extremely plastic and activate endogenous neural stem cells' self repair become the most potential treatment.As the recently study,the proliferation and differentiation of neural stem cells mainly depend on the change of its niche and the angiogenesis may be the foundation of neurogenesis and its niche.On account of VEGF contacting angiogenesis and neurogenesis,we suppose that vascular endothelial cells play an important role in the stem cell niche.In order to investigate the relationship between angiogenesis and the activation of endogenous neural stem cells in premature subventricular zone,we used adenovirus gene transfection and siRNA in vivo.After knocking-in and knocking-down VEGF,we observed the relationship between angiogenesis and the proliferation and differentiation of neural stem cells.[Method]we construct the VEGF adenovirus vector(Ad5-VEGF165-mCMV- EGFP) and VEGF-siRNA injecting through lateral cerebral and observed cellular changes in the subventricular zone(SVZ).of 3-day-old rats(approximately equivalent to premature infants) using immunofluorescence assays,Western blot analysis,and real-time quantitative PCR methods.Furthermore,we detect the level of VEGF in SVZ,observe the condition of angiogenesis and the proliferation and differentiation of neural stem cells,and analyze the relationship between them.[Result](1) Afer intracerebroventricular injecting VEGF adenovirus vector and VEGF-siRNA,VEGF was correspondingly up-regulation and down-regulation:In the present study,we used western blot analysis and real-time quantitative PCR methods to detect the level of VEGF after intracerebroventricular injecting,.The results showed that VEGF mRNA level was increased after injecting VEGF adenovirus vector and decreased after injecting VEGF-siRNA with statistical significant difference in 7 d.(2) Angiogenesis was promoted and inhibited corresponding with the change of VEGF level in SVZ;The angiogenesis and the proliferation and differentiation of neural stem cells were synchronism during period and space in SVZ: In order to observe the angiogenesis corresponding with the change of VEGF level in SVZ,we used immunofluorescence assays.The results showed that the area of vWF immuno-positive cells was increased after VEGF up-regulation and decreased after VEGF down- regulation with statistical significant difference in 7 d.We observe the relationship between the angiogenesis and the proliferation and differentiation of neural stem cells using the immunofluorescence assays.The results showed that vWF+cells and Nestin+ cells were simultaneously increased after VEGF up-regulation and decreased after VEGF down-regulation with statistical significant difference in 7 d.Nestin+ cells distributed around the vWF+ cells in SVZ.(3) The proliferation and differentiation of neural stem cells was promoted and inhibited corresponding with the change of angiogenesis in SVZ:In order to observe the proliferation and differentiation of neural stem cells was promoted and inhibited corresponding with the change of angiogenesis in SVZ,we used immunofluorescence assays,Western blot analysis,and real-time quantitative PCR methods.The results showed that:1) Nestin mRNA and protein level were increased after VEGF up-regulation and decreased after VEGF down-regulation with statistical significant difference in 7 d.In SVZ,Nestin+ cells and Nestin+/PCNA+ cells were increased after VEGF up-regulation and decreased after VEGF down-regulation with statistical significant difference in 7 d.2) Tuj1,GFAP,04 mRNA were increased aider VEGF up-regulation and decreased after VEGF down-regulation with statistical significant difference in 7 d.Map2+,GFAP+, 04+cells and Map2+/PCNA+,GFAP+/PCNA+,O4+/PCNA+ cells were increased after VEGF up-regulation and decreased after VEGF down-regulation with statistical significant difference in 7 d.(4)In cortex,the differentiation of neural stem cells was promoted and inhibited corresponding with the change of angiogenesis:we used immunofluorescence assays.The results showed that the area of Map2+,GFAP+,O4+ cells was increased after VEGF up-regulation and decreased after VEGF down-regulation with statistical significant difference in 7 d.[Conclusion]The angiogenesis and the proliferation and differentiation of neural stem cells were synchronism during period and space in SVZ.The neural stem cells distributed around the blood vessel.These provide clues for the dependablity of the angiogenesis and neurogenesis.The angiogenesis can promote the proliferation of neural stem cells,and enhanced the difference and migration of neural stem cells.The angiogenesis may play an important role in the period of neurogenesis,and provide a new way for preventing the brain injury of premature infant.