摘要
目的探讨缺氧诱导因子-1α(HIF-1α)及其靶基因血管内皮细胞生长因子(VEGF)在神经元缺氧缺血(HI)后的信号调控机制。方法将妊娠16~18dSD大鼠胎鼠的大脑皮层神经元进行原代培养,取培养5d的神经元建立氧糖剥离(OGD)模型,模拟细胞HI。应用磷脂酰肌醇-3激酶/丝氨酸/苏氨酸蛋白激酶(PI3K/Akt)信号通路特异性抑制剂wortmannin及LY294002预处理神经元,再进行OGD处理,同时设立对照组。培养的神经元分为4组:OGD后再灌注4h组、二甲基亚砜(DMSO)对照组、wortmannin干预组及LY294002干预组。采用RT-PCR及免疫细胞化学方法分别测定神经元HIF-1α、VEGFmRNA及蛋白表达水平。结果OGD再灌注4h后,wortmannin干预组及LY294002干预组HIF-1αmRNA水平较OGD再灌注4h组及DMSO对照组明显下降,VEGFmRNA水平亦较OGD再灌注4h组及DMSO对照组下调;OGD再灌注4h组HIF-1αmRNA及VEGFmRNA与DMSO对照组比较差异无统计学意义;免疫细胞化学结果显示wortmannin干预组及LY294002干预组HIF-1α、VEGF蛋白表达低于OGD后再灌注4h组及DMSO对照组,差异均有统计学意义(Pa<0.05),细胞黄棕色着色减弱,阳性细胞数减少。结论抑制PI3K/Akt信号通路后,OGD神经元HIF-1α、VEGFmRNA及其蛋白表达均下降,提示该信号传导通路参与HI后神经元HIF-1α及VEGF信号通路调控。
Objective To explore the mechanism of signaling modulation of hypoxia-inducible factor-1α(HIF-1α)and its target gene vascular endothelial growth factor(VEGF)in cultured cortical neurons during hypoxia-ischemia(HI).Methods The embryonic cerebral cortical neurons of pregnant SD rats at 16-18 days of gestation were cultured in vitro,and the neurons after being cultured for 5 days were adopted to establish the model of oxygen and glucose deprivation(OGD)to imitate HI environment.Before OGD treatment,neurons were pretreated with phosphatidylinositol-3 kinase/Akt(PI3K/Akt)pathway inhibitor as wortmannin or LY294002.Meanwhile,dmethyl sulfoxide(DMSO)pretreated neurons were used as controls.Cultured neurons were divided into 4 groups:OGD and 4 h reperfusion group,DMSO control group,wortmannin intervention group and LY294002 intervention group.Reverse transcription-polymerase chain reaction and immunocytochemistry were performed respectively to examine the expression levels of HIF-1α,VEGF mRNA and their protein in neurons.Results After reperfusion for 4 h followed by OGD,in wortmannin intervention group and LY294002 intervention group,HIF-1α mRNA was significantly lower than that in OGD group and 4 h reperfusion group and DMSO control group,and the levels of VEGF mRNA in wortmannin intervention group and LY294002 intervention group were also decreased compared with those in OGD and 4 h reperfusion group and DMSO control group.There was no significant difference in HIF-1α or VEGF mRNA between OGD and 4 h reperfusion group and DMSO control group.For immunocytochemistry analysis,the expression levels of HIF-1α and VEGF proteins in wortmannin intervention group and LY294002 intervention group were lower than those in DMSO control group,and there was statistical significance(Pa0.05),showing a decrease in yellow and brown stains of cell and in the number of positive cell.Conclusions After inhibition of PI3K/Akt signaling pathway,the levels of HIF-1α and VEGF mRNA and its protein were both attenuated in OGD neurons,indicating that this pathway was involved in modulating HIF-1α and VEGF in cultured cortical neurons after HI.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2009年第24期1875-1877,共3页
Journal of Applied Clinical Pediatrics
基金
国家自然科学基金项目资助(30825039
30770748)