核因子κB在低氧诱导因子1α基因修饰神经干细胞上调血管内皮生长因子表达中的桥接作用

Bridging effect of nuclear factor kappa B on upregulation of vascular endothelial growth factor expression in hypoxia-inducible factor-1 alpha gene-modified neural stem cells

背景:很多研究发现,在脑缺氧损伤区域核因子κB与血管内皮生长因子表达呈正相关。因此,作者大胆假设,核因子κB是否处于低氧诱导因子1α上调血管内皮生长因子作用通路上并起桥接作用。目的:以低氧诱导因子1α修饰的神经干细胞为载体,观察核因子κB在低氧诱导因子1α上调血管内皮生长因子表达通路中的作用。设计、时间及地点:细胞学体外观察,于2008-03/12在佳木斯大学神经科学研究所完成。材料:新生24h内的Wistar大鼠,雌雄不拘。方法:扩增腺病毒载体低氧诱导因子1α-绿色荧光蛋白后转染神经干细胞,荧光检测神经干细胞中低氧诱导因子1α-绿色荧光蛋白及空载体Ad-绿色荧光蛋白表达,分别提取基因转染后神经干细胞、空载体转染神经干细胞、正常神经干细胞蛋白。然后低氧诱导因子1α基因修饰的神经干细胞中按50,150,300μmol/L浓度梯度加入核因子κB特异性抑制剂二硫氨基甲酸酞吡咯烷,WesternBlot法检测其中血管内皮生长因子的表达变化。主要观察指标:各组神经干细胞中低氧诱导因子1α、血管内皮生长因子和核因子κB的表达;给予梯浓度核因子κB特异性抑制剂后神经干细胞中血管内皮生长因子的表达。结果:腺病毒低氧诱导因子1α-绿色荧光蛋白转染神经干细胞后基因表达强弱与MOI及转染时间有关;转染低氧诱导因子1α-绿色荧光蛋白后的神经干细胞中低氧诱导因子1α、血管内皮生长因子和核因子κB的表达呈正相关;给予梯浓度核因子κB特异性抑制剂后腺病毒低氧诱导因子1α-绿色荧光蛋白修饰的神经干细胞中血管内皮生长因子的表达呈抑制剂浓度依赖性下调,各浓度组之间血管内皮生长因子表达差异有显著性意义(P<0.05～0.01)。结论:核因子κB位于低氧诱导因子1α上调血管内皮生长因子表达的信号通路上并起桥接作用。

BACKGROUND：Numerous studies have shown that nuclear factor-κB （NF-κB） was positively correlated with vascular endothelial growth factor （VEGF） in the cerebral hypoxia region.Thus,we assumed whether NF-κB in the pathway of hypoxia-inducible factor-1α （HIF-1α） upregulating VEGF and plays a bridge effect.OBJECTIVE：To study the effects of NF-κB in HIF-1α pathway of increasing expression of VEGF using HIF-1α-modified neural stem cells （NSCs） as vectors.DESIGN,TIME AND SETTING：In vitro observation of cytology was conducted at the Neuroscience Institute,Jiamusi University from March to December 2008.MATERIALS：Wistar rats aged less than 24 hours of both genders were used.METHODS：NSCs were transfected after amplification of adenovirus vector HIF-1α （AdHIF-1α）-green fluorescent protein （GFP）.Fluorescence detection was used to determine HIF-1α-GFP and blank vector Ad-GFP expression in NSCs.Protein was extracted from transfected NSCs,blank vector NSCs and normal NSCs,separately.Subsequently,NF-κB specific inhibitor pyrrolidine dithiocarbamate （PDTC） （50,150,300 μmol/L） was added in HIF-1α-modified NSCs.Western blot analysis was used to determine changes in VEGF expression.MAIN OUTCOME MEASURES：The following parameters were measured：expression of HIF-1α,VEGF and NF-κB in NSCs in each group;VEGF expression in NSCs following treatment of NF-κB specific inhibitor.RESULTS：Gene expression was associated with MOI and transfected time following AdHIF-1α-GFP transfected with NSCs.After transfected AdHIF-1α-GFP in NSCs,HIF-1α,VEGF and NF-κB expression was positively correlated.Expression of VEGF was reduced in AdHIF-1α-GFP-modified NSCs following treatment of NF-κB inhibitor PDTC in a concentration-dependent fashion.There were significant differences in VEGF expression between each concentration group （P〈0.05-0.01）.CONCLUSION：NF-κB in signaling pathway of HIF-1α-upregulated VEGF expression and played a bridging effect.