雷帕霉素对正常和癫痫大鼠S6蛋白磷酸化的矛盾作用

发布时间：2018-04-22 05:07

本文选题：雷帕霉素 + mTOR信号通路　；参考：《浙江大学》2014年硕士论文

【摘要】：目的：红藻氨酸(KA)诱导的癫痫发作可激活哺乳动物的mammalian target of rapamycin (mTOR)信号通路作用靶点使下游S6蛋白磷酸化增加,mTOR抑制剂雷帕霉素可以抑制mTOR的激活,且可能有后续的潜在抗癫痫作用。然而,初步研究表明在KA注射前短时间内给予雷帕霉素,S6磷酸化反而引起mTOR信号通路的激活。在本研究中,我们同时在正常大鼠和KA癫痫模型大鼠进行了更详细地研究,探讨雷帕霉素这一矛盾性作用的规律。方法：正常组大鼠或KA模型组大鼠提前在不同时间点给予雷帕霉素,在给予雷帕霉素或癫痫发作后的不同时间点(1,3,6,10,15和24h)处死提取脑组织蛋白,采用Western Blotting(?)对mTOR信号靶点Akt, mTOR, Rictor, Raptor, S6K和S6的蛋白磷酸化进行了分析。对癫痫发作行为活动进行行为学监测,并根据改良的Racine法进行分级(每个时间点n=6)。使用Fluoro-Jade B染色检测神经细胞的死亡结果：我们发现在正常大鼠中,在注射雷帕霉素的3h-24h后,其表现出预期的对S6磷酸化的剂量依赖性抑制,然而在给予雷帕霉素后1h则发现了S6磷酸化水平反常的升高。在KA造模组大鼠中,在造模前10h以上使用雷帕霉素进行预处理可抑制KA癫痫引起的mTOR激活,相比之下,在KA造模前1h-6h使用雷帕霉素则会反常地加剧KA癫痫引起的mTOR激活。与对照组相比,在KA造模前1h使用雷帕霉素进行预处理的大鼠,癫痫发作更严重、持续时间更长,且神经细胞死亡数增加。而在KA造模前10h以上使用雷帕霉素进行预处理则对癫痫没有影响且能减少神经细胞的死亡。雷帕霉素对S6磷酸化的这种反常效应与上游的mTOR信号的改变相一致,且这种效应可被哌立福辛(一种Akt抑制剂)逆转。意义：这些数据表明了雷帕霉素对S6蛋白磷酸化的调节及其对癫痫发作的复杂性作用。在临床应用雷帕霉素作为癫痫及其他神经内科的治疗用药中应充分考虑到该反常作用。
[Abstract]:Aim: epileptic seizures induced by kainic acid can activate the mammalian target of rapamycin mTOR-signaling pathway in mammals, and the downstream phosphorylation of S6 protein can be increased by rapamycin, a inhibitor of kainic acid, which can inhibit the activation of mTOR. And may have subsequent potential antiepileptic effects. However, preliminary studies have shown that the phosphorylation of rapamycin I S6 at short time before Ka injection leads to activation of mTOR signaling pathway. In this study, we studied the contradictory effect of rapamycin in both normal rats and Ka epileptic rats in more detail. Methods: rats in normal group or Ka model group were given rapamycin at different time points in advance, and brain tissue proteins were extracted at different time points of rapamycin or epileptic seizure at different time points. The protein phosphorylation of Akt, mTOR, Rictor, Raptor, S6K and S6 were analyzed. The behavior of epileptic seizures was monitored and graded according to the modified Racine method. Detection of nerve cell death by Fluoro-Jade B staining Results: we found that in normal rats after injection of rapamycin (3h-24h), it showed the expected dose-dependent inhibition of S6 phosphorylation, but the abnormal increase of S6 phosphorylation level was found at 1 h after administration of rapamycin. In Ka model group, pretreatment with rapamycin more than 10 hours before model making could inhibit the activation of mTOR induced by Ka epilepsy, compared with that of 1h-6h before Ka model making, the activation of mTOR induced by Ka epilepsy could be increased unnaturally. Compared with the control group, the rats pretreated with rapamycin at 1 hour before Ka model had more severe seizure, longer duration and more nerve cell death. Pretreatment with rapamycin 10 hours or more before Ka model had no effect on epilepsy and could reduce the death of nerve cells. This anomalous effect of rapamycin on S6 phosphorylation is consistent with the change of upstream mTOR signal and can be reversed by pirifampicin, a Akt inhibitor. Significance: these data suggest that rapamycin regulates the phosphorylation of S 6 protein and its complexity in epileptic seizures. This anomalous effect should be taken into account in the clinical application of rapamycin as a therapeutic drug for epilepsy and other neuromedical departments.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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