调控mTOR信号通路对癫痫的作用及其机制研究

发布时间：2018-05-08 08:34

本文选题：雷帕霉素 + mTOR信号通路　；参考：《浙江大学》2016年博士论文

【摘要】：mTOR(mammalian target of rapamycn)信号通路是一条调节蛋白质合成、细胞生长、增殖等的信号通路。以往研究表明,mTOR信号通路参与了红藻氨酸(Kainic Acid,KA)诱导的大鼠癫痫的发生,并导致一系列癫痫后病理变化。雷帕霉素(Rapamycin)是mTOR信号通路特异性抑制剂,能够抑制mTOR信号通路的异常激活,并改善癫痫后病理变化以及减少自发性癫痫的产生,从而发挥潜在的抗癫痫作用。因此,在我们的研究中,我们探讨了雷帕霉素给药在正常SD大鼠以及KA诱发癫痫模型大鼠中对mTOR信号通路的作用。同时,构建条件性敲除神经前体细胞Rptor基因的小鼠,探讨敲除雷帕霉素作用靶点Raptor蛋白后对癫痫及并发症的影响,以明确mTOR信号通路在癫痫调控中的作用。第一章雷帕霉素对mTOR信号通路的矛盾性作用目的:前期研究发现,在KA诱导癫痫模型中提前较短时间给予雷帕霉素,更严重地激活以S6蛋白磷酸化为检测指标的mTOR信号通路,这与雷帕霉素对mTOR信号通路的抑制性作用相矛盾。因此,本研究拟在正常SD大鼠及KA诱导的SD大鼠癫痫模型中对雷帕霉素的矛盾性作用进行更详细的研究,明确雷帕霉素对mTOR信号通路及癫痫的作用。方法::在正常SD大鼠中给予单次不同剂量(0.3,1,3,1Omg/kg)的雷帕霉素以及给予单次单一剂量雷帕霉素(3mg/kg)作用不同时间点(1,3,6,15,24h),以确定雷帕霉素的量效与时效关系。同时,在KA诱导急性癫痫发作前,不同时间(1,3,6,15,24h)给予雷帕霉素以及雷帕霉素给药后急性癫痫发作不同时间(1,3,6,15,24h),检测S6蛋白磷酸化,以探讨雷帕霉素对癫痫mTOR信号通路的作用。另外,监测雷帕霉素不同时间(1,10h)给药后癫痫症状以及癫痫后神经细胞死亡,探讨雷帕霉素矛盾性作用的影响。最后,检测mTOR信号通路中Akt,Raptor,Rictor,S6k,S6蛋白磷酸化,以及给予Akt抑制剂哌立福辛(Perifosine),探讨雷帕霉素矛盾性作用的可能机制。结果:1.在正常SD大鼠中,提前3-24小时给予雷帕霉素能够抑制S6蛋白磷酸化,并呈剂量依赖性,而提前1小时给予雷帕霉素反而矛盾性增加了 S6蛋白磷酸。2.在KA诱导的急性癫痫模型中,提前10小时给予雷帕霉素,能够抑制癫痫导致mTOR信号通路的激活,而提前1-6小时给予雷帕霉素,反而矛盾性加重了癫痫导致mTOR信号通路的激活。3.提前1小时给予雷帕霉素,加重了癫痫的严重程度、癫痫持续时间及神经细胞死亡,而提前10小时给予雷帕霉素,对癫痫症状没有影响,但是可以减少神经元死亡。4.雷帕霉素对S6蛋白磷酸化的矛盾性作用与上游mTOR信号通路相关,并且,提前给予Akt抑制剂哌立福辛能够逆转雷帕霉素对S6磷酸化的矛盾性作用。结论:短时间给予雷帕霉素诱导了 S6蛋白激活,废除了雷帕霉素的抗癫痫作用。雷帕霉素对mTOR通路的调节具有高度复杂性,其原因可能与上游信号通路相关。第二章选择性敲除神经前体细胞中Rptor基因敲除对mTOR信号通路的作用目的:Raptor蛋白是mTOR信号通路中mTOR激酶调节相关蛋白,由Rptor基因编码。前期研究提示我们mTOR信号通路在癫痫发生中具有重要作用。为了探讨Raptor蛋白在癫痫及癫痫导致的相关疾病中的作用,我们制备在神经前体细胞中敲除Rptor的条件性敲除小鼠,以明确Raptor蛋白的作用。方法:构建条件性敲除神经前体细胞Rptor基因小鼠,对基因敲除小鼠外观及发育进行观察。同时,观察在正常情况及红藻氨酸(Kainic acid,KA)致痫后,基因敲除小鼠mTOR信号通路的改变及癫痫状态。FJB染色和TIMM染色分别观察KA致痫后基因敲除小鼠脑神经细胞死亡情况和苔藓纤维发芽情况,并EEG记录致痫后基因敲除小鼠自发性癫痫频率。最后,水迷宫实验,旷场实验和兴奋性实验观察小鼠行为学变化。结果:1.Rptor基因敲除小鼠(Rptor CKO mice)在外观与对照小鼠没有区别,并且Rptor基因敲除后,mTORC1信号通路被抑制。2.Rptor基因敲除小鼠脑重及体重比对照小鼠低,并且大脑上皮层神经元厚度减少。3.KA诱导癫痫后,KA导致的mTOR信号通路过度激活得到抑制,但Rptor基因敲除小鼠癫痫状态与对照小鼠没有明显差异。4.在Rptor基因敲除小鼠中,存在极少次数的自发性癫痫,且癫痫导致的苔藓纤维发芽得到改善。5.Rptor基因敲除小鼠在一定程度能够减少KA导致的认知障碍,减少焦虑行为及过度兴奋性。结论:尽管在神经前体细胞细胞中敲除了Rpto 基因后使小鼠早期发育受到影响,但在癫痫并发症及癫痫后行为中都得到一定改善。同时,也提示我们Raptor蛋白在癫痫及mTOR信号通路中有非常重要的作用。
[Abstract]:MTOR (mammalian target of rapamycn) signal pathway is a signal pathway that regulates protein synthesis, cell growth and proliferation. Previous studies have shown that mTOR signaling pathway participates in the occurrence of kainic acid (Kainic Acid, KA) induced rat epilepsy and causes a series of post epileptic pathological changes. Rapamycin (Rapamycin) is a mTOR signaling pathway. Road specific inhibitors can inhibit abnormal activation of mTOR signaling pathway, improve post epileptic pathological changes and reduce spontaneous epilepsy, and thus play a potential antiepileptic effect. Therefore, in our study, we explored the mTOR signal of rapamycin in normal SD rats and rats induced by KA induced epilepsy model. The role of the pathway. At the same time, a conditioned mouse knockout of the Rptor gene of the neural precursor cells was constructed to explore the effects of the knockout of the target Raptor protein on the epilepsy and the complications in order to clarify the role of the mTOR signaling pathway in the regulation of epilepsy. At present, rapamycin is given in the KA induced epileptic model in a shorter time and more seriously activates the mTOR signaling pathway with the S6 protein phosphorylation as the detection index, which is contradictory to the inhibitory effect of rapamycin on the mTOR signaling pathway. Therefore, this study is intended to be used in the normal SD rats and the KA induced SD rat model of the epileptic model of rapamycin. The effect of rapamycin on mTOR signaling pathway and epilepsy was studied in a more detailed study. Methods: in normal SD rats, a single dose of rapamycin (0.3,1,3,1Omg/kg) and a single single dose of rapamycin (3mg/kg) were given at different time points (1,3,6,15,24h) to determine the dose effect and time of rapamycin. At the same time, before KA induced acute epileptic seizures, different time (1,3,6,15,24h) was given to the acute seizures of rapamycin and rapamycin at different time (1,3,6,15,24h), and S6 protein phosphorylation was detected to explore the effect of rapamycin on the epileptic mTOR signaling pathway. In addition, the monitoring of rapamycin at different times (1,10h) was administered. The effects of rapamycin on the paradoxical effects of rapamycin were investigated. Finally, the possible mechanism of the paradoxical effect of rapamycin was explored in the mTOR signaling pathway Akt, Raptor, Rictor, S6k, S6 protein phosphorylation and Akt inhibitor piperinine (Perifosine). Results: 1. in normal SD rats, 3-24 Rapamycin was given to inhibit the phosphorylation of S6 protein in a dose dependent manner, while rapamycin was given 1 hours earlier, paradoxically increasing the S6 protein phosphoric acid.2. in the KA induced acute epilepsy model, and giving rapamycin 10 hours ahead of time, which could inhibit the activation of the mTOR signaling pathway by epilepsy and give thunder 1-6 hours ahead of time. Paradoxically, the paradoxical aggravation of epilepsy caused the activation of the mTOR signaling pathway to the activation of.3. 1 hours ahead of rapamycin, aggravating the severity of the epilepsy, the duration of epilepsy and the death of the nerve cells, and giving rapamycin 10 hours earlier, which did not affect the symptoms of epilepsy, but could reduce the neuronal death of.4. rapamycin to S6 eggs. The contradictory effect of white phosphorus acidification is related to the upstream mTOR signaling pathway, and the early administration of the Akt inhibitor piperinine can reverse the contradictory effect of rapamycin on S6 phosphorylation. Conclusion: short time rapamycin induced the activation of S6 protein and the antiepileptic effect of rapamycin. The regulation of rapamycin on the mTOR pathway There is a high degree of complexity, which may be associated with the upstream signal pathway. Second chapter second selectively knocks off the mTOR signaling pathway in the neural precursor cells: the Raptor protein is the mTOR kinase regulation related protein in the mTOR signaling pathway, and the Rptor gene is encoded. Earlier studies suggest that our mTOR signaling pathway is in epilepsy. In order to explore the role of Raptor protein in epilepsy and epilepsy related diseases, we prepare Rptor conditioned knockout mice in neural precursor cells to clarify the role of Raptor protein. Methods: to construct a conditioned knockout neural precursor cell Rptor gene mouse, and to develop the appearance and hair of the gene knockout mice. At the same time, we observed the changes in the mTOR signaling pathway in the gene knockout mice and the.FJB staining and TIMM staining in the epileptic state after epilepsy in normal conditions and Kainic acid (KA), and in the epileptic state.FJB staining and TIMM staining, respectively, to observe the brain cell death and the moss fiber germination of the gene knockout mice after KA induced epilepsy, and EEG to record the gene knockout after epilepsy. The frequency of spontaneous epileptic rats. Finally, the water maze test, open field experiment and excitatory experiment were used to observe the behavioral changes in mice. Results: the appearance of 1.Rptor gene knockout mice (Rptor CKO mice) was not different from that of the control mice, and the mTORC1 signaling pathway was suppressed by the mTORC1 signaling pathway and the brain weight and weight of the.2.Rptor knockout mice were smaller than those of the control. Rats were low, and the thickness of the neurons in the epithelial layer of the brain was reduced by.3.KA induced epilepsy. The overactivation of the mTOR signaling pathway caused by KA was inhibited, but there was no significant difference between the epileptic state of the Rptor knockout mice and the control mice. In the Rptor gene knockout mice, there were few spontaneous epileptic seizures, and the moss fiber germinated by epilepsy. The improvement of.5.Rptor gene knockout mice to some extent can reduce the cognitive impairment caused by KA and reduce anxiety behavior and excitability. Conclusion: Although the knockout of the Rpto gene in the neurons of the neural progenitor cells has been affected by the early development of the mice, there are some improvements in the epileptic complications and after epilepsy. It also suggests that Raptor protein plays a very important role in epilepsy and mTOR signaling pathway.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R965

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