【摘要】 目的 探讨低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)是否参与缺糖缺氧诱导的原代皮质神经元necroptosis。方法 原代皮质神经元培养14天,予caspase抑制剂Z-VAD-FMK(20μmol/L)预保护30 min,缺糖缺氧2 h,再灌注0、2、6、12、24、48 h,进行LDH测定;RT-PCR测HIF-1α RNA表达;Western blot检测总HIF-1α蛋白表达情况;分别提取细胞质和细胞核蛋白,Western blot分别检测胞质、胞核内HIF-1α蛋白表达情况。结果 caspase抑制剂Z-VAD-FMK预作用30 min、缺糖缺氧2 h、再灌注2 h后培养基中LDH增加(P<0.05),再灌注12 h达高峰;缺糖缺氧后HIF-1α RNA表达无变化(P>0.05);HIF-1α总蛋白表达增加(P<0.05),再灌注12 h达高峰;再灌注6 h时细胞质HIF-1α蛋白表达达高峰(P<0.05),随后降低;再灌注12 h时,细胞核HIF-1α蛋白表达达高峰(P<0.05),随后降低。结论 HIF-1α参与缺糖缺氧诱导的原代皮质神经元necroptosis。更多还原

【Abstract】 Objective To investigate whether hypoxia-inducible factor-1α(HIF-1α) is involved in primary cortical neuronal necroptosis induced by oxygen-glucose deprivation(OGD).Methods Primary cerebrocortical neurons were cultured for 14 days,followed by pretreatment with Z-VAD-FMK(20 μmol/L) for 30 min prior to exposure to OGD for 2 hours(h) and reoxygenation for different durations(0,2,6,12,24 and 48 hours).Cell viability was determined by determination of LDH level;RT-PCR was employed to determine the expression of HIF-1α RNA,and the expression of total proteins of HIF-1α was determined by Western blot analysis;the cytoplasmic and nuclear proteins of HIF-1α were respectively extracted for the determination of cytoplasmic and nuclear protein expression levels by Western blot.Results There was an increase in LDH level in the culture medium following 30-minute pretreatment with caspase inhibitor Z-VAD-FMK,exposure to OGD for 2 h and reperfusion for 2 h(P <0.05),with its peak at 12h of reperfusion;there was no change in the expression of HIF-1α RNA subsequent to OGD(P> 0.05);the expression of total proteins of HIF-1α was upregulated(P <0.05),with its peak at 12 h of reperfusion;the protein expression of cytoplasmic HIF-1α reached its peak at 6 h of reperfusion(P <0.05) and declined thereafter;the protein expression of nuclear HIF-1α reached its peak at 12 h of reperfusion(P <0.05) and declined afterwards.Conclution HIF-1αwas involved in oxygen-glucose deprivation-induced primary cortical neuronal necroptosis.更多还原