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目的探讨缺氧诱导因子1α(hypoxia-inducible factor1α,HIF-1α)及细胞外调节蛋白激酶1/2(extra-cellular signal-regulated kinase1/2,ERK1/2)在体外培养的胎鼠大脑皮质神经元缺氧缺血再灌注后变化的趋势及其相互关系。方法取15只16～18d孕龄SD大鼠的大脑皮层,进行皮质神经元原代培养,取培养5d的原代神经元建立氧糖剥离(oxygen and glucose deprivation,OGD)模型。实验分为实验组1:OGD模型制备后,更换培养液为含葡萄糖的神经元完全培养基,以形成再灌注;取再灌注0、2、4、8、12、24h观察;对照组1:正常培养液孵育的神经元;实验组2:U0126预处理神经元后制备OGD模型,于再灌注后4、8h观察;对照组2:DMSO预处理神经元后制备OGD模型,余同实验组2。应用Westernblot定量测定各组HIF-1α、VEGF蛋白及ERK1/2、p-ERK1/2的表达变化,SABC免疫细胞化学法检测p-ERK1/2、HIF-1α蛋白表达及分布情况。结果培养5d的皮质神经元突起间互相连接成网状。实验组1HIF-1α、VEGF蛋白表达逐渐升高,8h表达量最高,12h后逐渐下降,与对照组1比较差异有统计学意义(P<0.05)。各时间点实验组1与对照组1ERK1/2蛋白表达无明显差异(P>0.05)。实验组1p-ERK1/2蛋白表达在2h时开始增加,4h达高峰,8h开始下降,与对照组1比较差异有统计学意义(P<0.01);实验组2p-ERK1/2蛋白表达下降,HIF-1α及VEGF蛋白表达也随之下调,与对照组2比较差异均有统计学意义(P<0.05)。各时间点实验组2和对照组2ERK1/2蛋白表达差异无统计学意义(P>0.05)。免疫细胞化学染色显示神经元黄棕色着色减弱,p-ERK1/2、HIF-1α表达下降。结论HIF-1α及其靶基因VEGF蛋白在OGD再灌注后的皮质神经元表达具有一定时间规律性,抑制ERK1/2信号通路后这两个蛋白表达均下降,提示该通路在神经元OGD后诱导HIF-1α及VEGF的调控中起重要作用。
Objective To investigate the effects of hypoxia-inducible factor 1α (HIF-1α) and extracellular signal-regulated kinase1 / 2 (ERK1 / 2) on cultured rat cerebral cortical neurons The trend of changes after hypoxia - ischemia / reperfusion and its relationship. Methods 15 cortical neurons were cultured in the cerebral cortex of 15 SD rats aged 16-18 days. Oxygen and glucose deprivation (OGD) models were established by culturing primary cortical neurons for 5 days. The experiment was divided into experimental group 1: After the OGD model was prepared, the medium was replaced with glucose-containing neurons complete medium to form reperfusion; reperfusion 0,2,4,8,12,24h; control group 1: OGD model was prepared after U0126 preconditioning neurons in experiment group 2 and 4 and 8 hours after reperfusion. OGD model was prepared in control group 2: DMSO preconditioning neurons, the same experimental group 2 . The expression of HIF-1α, VEGF protein and ERK1 / 2 and p-ERK1 / 2 in each group were quantified by Western blot. The protein expression and distribution of p-ERK1 / 2 and HIF-1α were detected by SABC immunocytochemistry. Results The cortical neurons cultured for 5 days were connected to each other into a network. The expression of 1HIF-1αand VEGF protein in the experimental group increased gradually, reached the peak at 8h, then decreased gradually after 12h, which was significantly different from that in control group 1 (P <0.05). There was no significant difference in the expression of 1ERK1 / 2 between experimental group 1 and control group at each time point (P> 0.05). The expression of 1p-ERK1 / 2 in experimental group began to increase at 2h, peaked at 4h, and then decreased at 8h, which was significantly different from that in control group 1 (P <0.01). The protein expression of 2p-ERK1 / The expression of HIF-1α and VEGF also decreased, which was significantly different from that in control group 2 (P <0.05). There was no significant difference in the expression of 2ERK1 / 2 between experimental group 2 and control group at each time point (P> 0.05). Immunocytochemical staining showed that the neuronal yellow-brown coloration was weakened and the expression of p-ERK1 / 2 and HIF-1α was decreased. Conclusion The expression of HIF-1α and its target gene VEGF protein in the cortical neurons after OGD reperfusion has a certain regularity of time, and the expression of these two proteins is decreased after inhibiting the ERK1 / 2 signaling pathway, suggesting that this pathway is induced after OGD of neurons HIF-1α and VEGF play an important role in the regulation.