癫痫持续状态大鼠海马内Notch通路对HIF-1α调控位点的研究

摘要

目的：研究低氧诱导因子-1α(HIF-1α)及Notch信号通路下游基因HES 1在发育期癫痫持续状态(SE)大鼠海马内的表达变化，探寻Notch信号通路对于HIF-1α的调控位点。方法176只21日龄SD大鼠随机分为生理盐水对照组(NS组)、戊四氮(PTZ)致SE模型组(PTZ组)、Notch信号通路特异性抑制剂(DAPT)干预组(DAPT组)。PTZ组大鼠腹腔注射PTZ溶液制作SE模型，NS组注射等剂量的生理盐水作为对照，予安定腹腔注射终止SE发作。造模成功后分别于0.5、1、2、4、8 h断头取脑分离双侧海马组织，采用RT-PCR法检测HES 1、HIF-1αmRNA的表达；于2、4、8、12、24 h断头取海马，采用Western blot法检测蛋白的表达。DAPT组在开始造模前30 min腹腔注射DAPT溶液，分别于造模成功后2 h、8 h断头取海马组织，采用RT-PCR法检测2 h时HES 1、HIF-1αmRNA的表达，用Western blot法检测8 h时蛋白的表达。结果 SE后的各个时间点，大鼠海马内HES1、HIF-1αmRNA和蛋白表达是增高的，与同一时间的NS组相比差异有统计学意义(P< 0.05)；与同一时间点的PTZ组比较，DAPT组的HES 1、HIF-1α mRNA和蛋白表达明显降低，差异有统计学意义(P< 0.05)。结论在发育期大鼠SE发作后的海马内，HES 1基因可能是Notch信号通路中对HIF-1α表达的调控位点。%Objective To study the expression of hypoxia inducible factor-1 (HIF-1 ) and Notch signaling pathway downstream gene HES 1 in the hippocampus of pubertal rats with status epilepsy (SE), and to explore the regulation site of HIF-1αin Notch signaling pathway. Methods One hundred and seventy-six 21-day-old SD rats were randomly divided into control group (NS group), pentetrazole (PTZ)-induced SE group (PTZ group), and Notch signaling pathway speciifc inhibitor (DAPT) intervention group (DAPT group). In PTZ group PTZ was intraperitoneally injected to build SE model and in NS group normal saline was injected as control. The intraperitoneal injection of diazepam was used to terminate SE seizures. After successful modeling, the bilateral hippocampuses were isolated after the rats were sacriifced at 0.5, 1, 2, 4 and 8 h, respectively, and RT-PCR was performed to detect the mRNA expression of HES 1 and HIF-1α. The Western Blot was performed to detect protein expression in hippocampuses which were collected at 2 , 4 , 8 , 12 , and 24 h after successful modeling. DAPT group received intraperitoneal injection of DAPT 30 min before the start of molding, then the hippocampuses were isolated at 2 and 8 h after successful modeling. RT-PCR was performed to detect the mRNA expression of HES 1 and HIF-1αat 2 h, and Western blot was performed to detect protein expression at 8 h. Results At each time point after SE, the expression of mRNA of HES 1 and HIF-1αand the expression of protein were higher than the same time point of NS group (P< 0 . 05 ). Compared with the same time point of PTZ group, the mRNA expression of HES 1 and HIF-1αand the expression of protein of DAPT group were obviously reduced (P< 0 . 05 ). Conclusion HES 1 gene may be the regulatory site of HIF-1 expression in Notch signaling pathway in the hippocampus of puberty rats with SE.