毛冬青甲素对脑缺血后Notch通路的调控作用与神经元再生

发布时间：2018-03-14 03:48

  本文选题：毛冬青甲素　切入点：局灶性脑缺血再灌注　出处：《福建医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究毛冬青甲素（Ilexonin A，IA）对大鼠脑缺血再灌注后缺血周边区Notch信号的调节作用与神经干细胞增殖和分化的关系，，探讨脑损伤后的修复机制。 方法:线栓左侧大脑中动脉阻塞（middle cerebral artery occlusion，MCAO）2h，制备脑缺血再灌注损伤模型。 SD大鼠随机分为：正常组、假手术组、模型组、IA组，其中模型组、IA组（IA40mg/kg）分别再灌注不同时间。神经功能学评分评估大鼠的神经功能恢复情况；TTC染色验证实验动物模型和提供定位标记；免疫荧光法观察缺血周边区增值细胞标记物Brdu、神经干细胞标志物巢蛋白（Nestin）共表达的阳性细胞数，Brdu、神经元标志物神经元核蛋白（Neuronalnucleus protein,Neun）共表达的阳性细胞数及Notch信号通路分子Notch信号胞内域(Notch intracellular domain,NICD)的阳性细胞数，蛋白印迹法检测NICD的表达情况；RT-PCR法检测Notch信号通路中Notch1受体蛋白、早老蛋白1（Presenilin,PS1）、靶基因HES1的表达情况。 结果：（1）神经功能恢复情况：模型组和IA组脑缺血再灌注1天时神经功能缺损评分明显增高，随着时间的增加评分逐渐降低，并且IA组与模型组在3天、7天、14天降低显著（p0.01、p0.05）。（2）缺血周边区神经再生情况：IA组与模型组在再灌注第3天相比较，IA组缺血周边区Brdu/Nestin阳性细胞数增殖明显（p0.05），7天时表达的细胞数最多（p0.05）。再灌注第14天，IA组和模型组Brdu/Neun阳性细胞仅有少量表达且无显著性意义。（3）Notch信号通路的激活和各基因的表达情况：模型组和IA组与正常组和假手术组比较，在再灌注1天NICD表达增加，且在1天、3天、7天均增高（p0.05）。模型组、IA组Notch信号通路分子Notch1, PS1, HES1的表达较正常组和假手术组均升高，IA组与模型组比较在3天，7天时表达增加（p0.01）。 结论 1. IA在脑缺血再灌注后促进大鼠神经功能的恢复，其机制与IA促进缺血周边区Nestin阳性细胞的增殖有关。 2. IA在脑缺血再灌注后早期对Notch信号通路各分子Notch1、NICD、PS1、HES1的表达具有促进作用，提示脑缺血再灌注后早期IA促进Notch信号通路的激活。
[Abstract]:Aim: to study the effect of Ilexonin A (Ilexonin A) on the regulation of Notch signal and the proliferation and differentiation of neural stem cells (NSCs) after cerebral ischemia-reperfusion in rats, and to explore the repair mechanism after brain injury. Methods: the middle cerebral artery occlusion of left middle cerebral artery (MCAO) was established for 2 h. SD rats were randomly divided into normal group, sham operation group, model group and IA group. The model group (IA group) was perfused with 40 mg / kg IA for different time after reperfusion. The neurological function score was used to evaluate the recovery of neural function in rats. TTC staining was used to verify the experimental animal model and to provide localization markers. The number of positive cells co-expressed by Brdu, Nestin, a neural stem cell marker, and the number of positive cells co-expressed in neuronal marker neuronal nuclear nucleus nestin by immunofluorescence assay and Notch signal transduction. The number of positive cells of Notch intracellular domain (NICD) in the intracellular domain of pathway molecule Notch signal, Western blot was used to detect the expression of NICD and RT-PCR was used to detect the expression of Notch1 receptor protein, presenilin PS1 protein and target gene HES1 in Notch signaling pathway. Results (1) recovery of neural function: the neurological deficit score of model group and IA group increased significantly after 1 day of cerebral ischemia reperfusion, and decreased gradually with the increase of time. Compared with model group on the 3rd day after reperfusion, the proliferation of Brdu/Nestin positive cells in ischemic peripheral area was significantly increased in group IA and model group at day 7 and day 7. The expression of Brdu/Nestin positive cells in ischemic peripheral area of group A was significantly higher than that in group A at 7 days after reperfusion (P < 0.05), and that in the model group was significantly lower than that in group A on the 3rd day after reperfusion (P < 0.05), and there was no significant difference in the expression of Brdu/Nestin positive cells between the control group and the model group. On the 14th day after reperfusion, the Brdu/Neun positive cells in the IA group and the model group had only a small amount of expression and no significant difference. The activation of Notch signaling pathway and the expression of the genes in the model group and IA group were compared with those in the normal group and the sham operation group. The expression of Notch signal pathway molecules Notch1, PS1 and HES1 in the model group was higher than that in the normal group and sham operation group. The expression of Notch signal pathway molecules in the model group was higher than that in the control group and sham operation group. Conclusion. 1. IA promoted the recovery of neural function in rats after cerebral ischemia-reperfusion, and its mechanism was related to the proliferation of Nestin positive cells in ischemic peripheral area. 2. IA promoted the expression of Notch1NICD1PS1HES1 in the Notch signal pathway early after cerebral ischemia-reperfusion, suggesting that IA promoted the activation of Notch signal pathway in the early stage of cerebral ischemia-reperfusion.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3


本文编号：1609468