發(fā)布時(shí)間：2018-01-05 03:02

  本文關(guān)鍵詞：PRAS40緩解神經(jīng)毒性多肽誘導(dǎo)神經(jīng)元凋亡機(jī)制的研究　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 凋亡 負(fù)反饋調(diào)節(jié)機(jī)制 PI3K-Akt-mTOR信號(hào)通路 PRAS40 朊病毒病

【摘要】：傳染性海綿狀腦病(Transmissible spongiform encephalopathies,TSEs)也被稱為朊病毒病,是一類具有傳染性、致死性的神經(jīng)退行性疾病。該疾病的原因通常是因?yàn)樗拗鳈C(jī)體內(nèi)正常的細(xì)胞型阮蛋白(Cellular Prion Protein,PrPc)發(fā)生錯(cuò)誤折疊后轉(zhuǎn)化為具有致病性的朊蛋白(Scrapie Prion Protein,PrPSc)。PrPSc具有部分蛋白酶抗性,并能夠誘導(dǎo)PrPC轉(zhuǎn)變成PrPSc,在體內(nèi)大量蓄積引起宿主發(fā)病。TSEs的病理學(xué)特點(diǎn)包括海綿狀病變、小膠質(zhì)細(xì)胞激活、星形膠質(zhì)細(xì)胞增生以及神經(jīng)元死亡。無法清除腦中致病性的PrPSc將導(dǎo)致神經(jīng)元的功能障礙。目前仍然沒有有效的應(yīng)對(duì)朊病毒病的治療方法以及預(yù)防控制措施。哺乳動(dòng)物雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)在細(xì)胞生長(zhǎng)以及新陳代謝中都具有重要的調(diào)節(jié)作用。許多研究都表明mTOR的活性與神經(jīng)退行性疾病的致病機(jī)制相關(guān)。PRAS40(proline-richAktsubstrateof40-kDa)是 mTORC1 的直接抑制蛋白,并且連接 AKT 和 mTOR通路。mTOR被認(rèn)為具有調(diào)節(jié)細(xì)胞生長(zhǎng)和新陳代謝的重要功能。許多研究表明,異常的mTOR活性與許多神經(jīng)退行性疾病引起的認(rèn)知障礙都有非常緊密的關(guān)系。本研究通過神經(jīng)毒性多肽PrP106-126,建立朊病毒病(即傳染性海綿狀腦病)的疾病模型,進(jìn)而研究朊病毒病中mTOR調(diào)節(jié)細(xì)胞凋亡的分子機(jī)制:(1)PrP106-126作用小鼠神經(jīng)瘤母細(xì)胞系(N2a),用免疫印跡法檢測(cè)mTOR磷酸化的水平。研究結(jié)果表明:mTOR的磷酸化水平隨時(shí)間的延長(zhǎng)而顯著升高。并且發(fā)現(xiàn),mTOR的激活是由于PrP106-126引起的ROS產(chǎn)生,通過加入ROS的抑制劑NAC,磷酸化的mTOR水平明顯降低。因此,PrP106-126處理后激活的mTOR通路與ROS的產(chǎn)生有一定關(guān)聯(lián)。(2)轉(zhuǎn)染HA-PRAS40的細(xì)胞用神經(jīng)毒性多肽PrP106-126處理,與轉(zhuǎn)染HA空白載體的對(duì)照組相比,過表達(dá)PRAS40的實(shí)驗(yàn)組細(xì)胞凋亡水平明顯下降。過表達(dá)PRAS40的細(xì)胞在經(jīng)過神經(jīng)毒性多肽PrP106-126處理后,與轉(zhuǎn)染空白載體的對(duì)照組相比,caspase-3剪切體和PARP剪切體的量明顯下降。上述結(jié)果表明,過表達(dá)PRAS40可以緩解神經(jīng)毒性多肽引起的神經(jīng)元凋亡。(3)在神經(jīng)毒性多肽PrP106-126處理后,與轉(zhuǎn)染空白載體的對(duì)照組相比,轉(zhuǎn)染了 HA-PRAS40的細(xì)胞表現(xiàn)出了 S6K1和4EBP1磷酸化水平的下降。mTOR信號(hào)通路在神經(jīng)毒性多肽PrP106-126作用后可以被過度活化,而這種活化可以通過PRAS40的過表達(dá)或者雷帕霉素的處理而被抑制。(4)在過表達(dá)PRAS40之后,Akt和GSK3 β的磷酸化水平明顯升高,表明PI3K-Akt信號(hào)通路被激活。加入Akt的抑制劑渥漫青霉素后,發(fā)現(xiàn)PRAS40對(duì)PrP106-126引起的神經(jīng)元凋亡的抑制作用被減弱了,因此,PRAS40緩解PrP106-126引起的神經(jīng)元凋亡,是通過恢復(fù)Akt的磷酸化活性來實(shí)現(xiàn)的。Akt可能部分參與了 mTOR調(diào)節(jié)的PrP106-126引起的神經(jīng)元凋亡。PRAS40抑制了 mTORC1的過度活化,并且在保護(hù)細(xì)胞應(yīng)對(duì)神經(jīng)毒性多肽引起的凋亡中發(fā)揮了重要作用。因而,PRAS40作為靶蛋白,可為Prion疾病的治療提供潛在的方法和思路。
[Abstract]:Transmissible spongiform encephalopathy (TSEs) is also known as prion disease. Is a class of infectious and fatal neurodegenerative diseases. The cause of the disease is usually due to the normal cellular Prion Protein of Ruan protein. PrPc) was converted into a pathogenicity prion protein Scrapie Prion protein PrPScn. PrPSc showed partial protease resistance. And can induce PrPC to transform into PrPSc.The pathological characteristics of host pathophysiology include spongy lesion and microglia activation. Astrocyte proliferation and neuronal death. Failure to remove pathogenetic PrPSc in the brain will lead to neuronal dysfunction. There is still no effective treatment for prion disease and prevention and control measures. Mammalian rapamycin target protein. Mammalian target of rapamycin. MTORs play an important role in cell growth and metabolism. Many studies have shown that the activity of mTOR is related to the pathogenesis of neurodegenerative diseases. Prorichline-Akt substrate of 40-kDa is a direct inhibitor of mTORC1. And linking the AKT and mTOR pathways. MTOR is thought to have an important role in regulating cell growth and metabolism. Many studies have shown that. Abnormal mTOR activity is closely related to cognitive impairment caused by many neurodegenerative diseases. This study is based on neurotoxic polypeptide PrP106-126. To establish a disease model of prion disease (i.e. infectious spongiform encephalopathy). Furthermore, the molecular mechanism of mTOR regulating apoptosis in prion disease was studied. The effect of PrP106-126 on mouse neuroma cell line (N2a) was studied. The level of phosphorylation of mTOR was detected by Western blotting. The results showed that the phosphorylation level of mTOR increased significantly with time. The activation of mTOR was due to the production of ROS induced by PrP106-126, and the level of phosphorylated mTOR was significantly decreased by adding the inhibitor of ROS. Activation of mTOR pathway after PrP106-126 treatment was associated with the production of ROS. The cells transfected with HA-PRAS40 were treated with neurotoxic polypeptide PrP106-126. Compared with the control group transfected with HA blank vector. The level of apoptosis in the experimental group with overexpression of PRAS40 was significantly decreased, and the cells with overexpression of PRAS40 were treated with neurotoxic polypeptide PrP106-126. Compared with the control group transfected with blank vector, the amount of caspase-3 and PARP shearing bodies were significantly decreased. Overexpression of PRAS40 can alleviate neuronal apoptosis induced by neurotoxic polypeptide. The cells transfected with HA-PRAS40 showed that. The decrease of phosphorylation level of S6K1 and 4EBP1. MTOR signaling pathway can be over-activated by neurotoxic polypeptide PrP106-126. This activation can be inhibited by overexpression of PRAS40 or treatment of rapamycin. (4) after overexpression of PRAS40, the phosphorylation levels of Akt and GSK3 尾 are significantly increased. The results indicated that the PI3K-Akt signaling pathway was activated. It was found that the inhibitory effect of PRAS40 on neuronal apoptosis induced by PrP106-126 was weakened, so PRAS40 alleviated the neuronal apoptosis induced by PrP106-126. This is achieved by restoring the phosphorylation activity of Akt. Akt may be partially involved in the apoptosis of neurons induced by PrP106-126 regulated by mTOR. PRAS40 inhibits neuronal apoptosis. Overexpression of mTORC1. It plays an important role in protecting cells from apoptosis induced by neurotoxic polypeptides. Therefore, as a target protein, PRAS40 can provide potential methods and ideas for the treatment of Prion disease.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S855.3

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