本文選題：雷帕霉素 + mTOR信號通路��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：mTOR(mammalian target of rapamycn)信號通路是一條調(diào)節(jié)蛋白質(zhì)合成、細(xì)胞生長、增殖等的信號通路。以往研究表明,mTOR信號通路參與了紅藻氨酸(Kainic Acid,KA)誘導(dǎo)的大鼠癲癇的發(fā)生,并導(dǎo)致一系列癲癇后病理變化。雷帕霉素(Rapamycin)是mTOR信號通路特異性抑制劑,能夠抑制mTOR信號通路的異常激活,并改善癲癇后病理變化以及減少自發(fā)性癲癇的產(chǎn)生,從而發(fā)揮潛在的抗癲癇作用。因此,在我們的研究中,我們探討了雷帕霉素給藥在正常SD大鼠以及KA誘發(fā)癲癇模型大鼠中對mTOR信號通路的作用。同時(shí),構(gòu)建條件性敲除神經(jīng)前體細(xì)胞Rptor基因的小鼠,探討敲除雷帕霉素作用靶點(diǎn)Raptor蛋白后對癲癇及并發(fā)癥的影響,以明確mTOR信號通路在癲癇調(diào)控中的作用。第一章雷帕霉素對mTOR信號通路的矛盾性作用目的:前期研究發(fā)現(xiàn),在KA誘導(dǎo)癲癇模型中提前較短時(shí)間給予雷帕霉素,更嚴(yán)重地激活以S6蛋白磷酸化為檢測指標(biāo)的mTOR信號通路,這與雷帕霉素對mTOR信號通路的抑制性作用相矛盾。因此,本研究擬在正常SD大鼠及KA誘導(dǎo)的SD大鼠癲癇模型中對雷帕霉素的矛盾性作用進(jìn)行更詳細(xì)的研究,明確雷帕霉素對mTOR信號通路及癲癇的作用。方法::在正常SD大鼠中給予單次不同劑量(0.3,1,3,1Omg/kg)的雷帕霉素以及給予單次單一劑量雷帕霉素(3mg/kg)作用不同時(shí)間點(diǎn)(1,3,6,15,24h),以確定雷帕霉素的量效與時(shí)效關(guān)系。同時(shí),在KA誘導(dǎo)急性癲癇發(fā)作前,不同時(shí)間(1,3,6,15,24h)給予雷帕霉素以及雷帕霉素給藥后急性癲癇發(fā)作不同時(shí)間(1,3,6,15,24h),檢測S6蛋白磷酸化,以探討雷帕霉素對癲癇mTOR信號通路的作用。另外,監(jiān)測雷帕霉素不同時(shí)間(1,10h)給藥后癲癇癥狀以及癲癇后神經(jīng)細(xì)胞死亡,探討雷帕霉素矛盾性作用的影響。最后,檢測mTOR信號通路中Akt,Raptor,Rictor,S6k,S6蛋白磷酸化,以及給予Akt抑制劑哌立福辛(Perifosine),探討雷帕霉素矛盾性作用的可能機(jī)制。結(jié)果:1.在正常SD大鼠中,提前3-24小時(shí)給予雷帕霉素能夠抑制S6蛋白磷酸化,并呈劑量依賴性,而提前1小時(shí)給予雷帕霉素反而矛盾性增加了 S6蛋白磷酸。2.在KA誘導(dǎo)的急性癲癇模型中,提前10小時(shí)給予雷帕霉素,能夠抑制癲癇導(dǎo)致mTOR信號通路的激活,而提前1-6小時(shí)給予雷帕霉素,反而矛盾性加重了癲癇導(dǎo)致mTOR信號通路的激活。3.提前1小時(shí)給予雷帕霉素,加重了癲癇的嚴(yán)重程度、癲癇持續(xù)時(shí)間及神經(jīng)細(xì)胞死亡,而提前10小時(shí)給予雷帕霉素,對癲癇癥狀沒有影響,但是可以減少神經(jīng)元死亡。4.雷帕霉素對S6蛋白磷酸化的矛盾性作用與上游mTOR信號通路相關(guān),并且,提前給予Akt抑制劑哌立福辛能夠逆轉(zhuǎn)雷帕霉素對S6磷酸化的矛盾性作用。結(jié)論:短時(shí)間給予雷帕霉素誘導(dǎo)了 S6蛋白激活,廢除了雷帕霉素的抗癲癇作用。雷帕霉素對mTOR通路的調(diào)節(jié)具有高度復(fù)雜性,其原因可能與上游信號通路相關(guān)。第二章選擇性敲除神經(jīng)前體細(xì)胞中Rptor基因敲除對mTOR信號通路的作用目的:Raptor蛋白是mTOR信號通路中mTOR激酶調(diào)節(jié)相關(guān)蛋白,由Rptor基因編碼。前期研究提示我們mTOR信號通路在癲癇發(fā)生中具有重要作用。為了探討Raptor蛋白在癲癇及癲癇導(dǎo)致的相關(guān)疾病中的作用,我們制備在神經(jīng)前體細(xì)胞中敲除Rptor的條件性敲除小鼠,以明確Raptor蛋白的作用。方法:構(gòu)建條件性敲除神經(jīng)前體細(xì)胞Rptor基因小鼠,對基因敲除小鼠外觀及發(fā)育進(jìn)行觀察。同時(shí),觀察在正常情況及紅藻氨酸(Kainic acid,KA)致癇后,基因敲除小鼠mTOR信號通路的改變及癲癇狀態(tài)。FJB染色和TIMM染色分別觀察KA致癇后基因敲除小鼠腦神經(jīng)細(xì)胞死亡情況和苔蘚纖維發(fā)芽情況,并EEG記錄致癇后基因敲除小鼠自發(fā)性癲癇頻率。最后,水迷宮實(shí)驗(yàn),曠場實(shí)驗(yàn)和興奮性實(shí)驗(yàn)觀察小鼠行為學(xué)變化。結(jié)果:1.Rptor基因敲除小鼠(Rptor CKO mice)在外觀與對照小鼠沒有區(qū)別,并且Rptor基因敲除后,mTORC1信號通路被抑制。2.Rptor基因敲除小鼠腦重及體重比對照小鼠低,并且大腦上皮層神經(jīng)元厚度減少。3.KA誘導(dǎo)癲癇后,KA導(dǎo)致的mTOR信號通路過度激活得到抑制,但Rptor基因敲除小鼠癲癇狀態(tài)與對照小鼠沒有明顯差異。4.在Rptor基因敲除小鼠中,存在極少次數(shù)的自發(fā)性癲癇,且癲癇導(dǎo)致的苔蘚纖維發(fā)芽得到改善。5.Rptor基因敲除小鼠在一定程度能夠減少KA導(dǎo)致的認(rèn)知障礙,減少焦慮行為及過度興奮性。結(jié)論:盡管在神經(jīng)前體細(xì)胞細(xì)胞中敲除了Rpto 基因后使小鼠早期發(fā)育受到影響,但在癲癇并發(fā)癥及癲癇后行為中都得到一定改善。同時(shí),也提示我們Raptor蛋白在癲癇及mTOR信號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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R965

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