PRAS40缓解神经毒性多肽诱导神经元凋亡机制的研究

发布时间：2018-01-05 03:02

本文关键词：PRAS40缓解神经毒性多肽诱导神经元凋亡机制的研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：传染性海绵状脑病(Transmissible spongiform encephalopathies,TSEs)也被称为朊病毒病,是一类具有传染性、致死性的神经退行性疾病。该疾病的原因通常是因为宿主机体内正常的细胞型阮蛋白(Cellular Prion Protein,PrPc)发生错误折叠后转化为具有致病性的朊蛋白(Scrapie Prion Protein,PrPSc)。PrPSc具有部分蛋白酶抗性,并能够诱导PrPC转变成PrPSc,在体内大量蓄积引起宿主发病。TSEs的病理学特点包括海绵状病变、小胶质细胞激活、星形胶质细胞增生以及神经元死亡。无法清除脑中致病性的PrPSc将导致神经元的功能障碍。目前仍然没有有效的应对朊病毒病的治疗方法以及预防控制措施。哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)在细胞生长以及新陈代谢中都具有重要的调节作用。许多研究都表明mTOR的活性与神经退行性疾病的致病机制相关。PRAS40(proline-richAktsubstrateof40-kDa)是 mTORC1 的直接抑制蛋白,并且连接 AKT 和 mTOR通路。mTOR被认为具有调节细胞生长和新陈代谢的重要功能。许多研究表明,异常的mTOR活性与许多神经退行性疾病引起的认知障碍都有非常紧密的关系。本研究通过神经毒性多肽PrP106-126,建立朊病毒病(即传染性海绵状脑病)的疾病模型,进而研究朊病毒病中mTOR调节细胞凋亡的分子机制:(1)PrP106-126作用小鼠神经瘤母细胞系(N2a),用免疫印迹法检测mTOR磷酸化的水平。研究结果表明:mTOR的磷酸化水平随时间的延长而显著升高。并且发现,mTOR的激活是由于PrP106-126引起的ROS产生,通过加入ROS的抑制剂NAC,磷酸化的mTOR水平明显降低。因此,PrP106-126处理后激活的mTOR通路与ROS的产生有一定关联。(2)转染HA-PRAS40的细胞用神经毒性多肽PrP106-126处理,与转染HA空白载体的对照组相比,过表达PRAS40的实验组细胞凋亡水平明显下降。过表达PRAS40的细胞在经过神经毒性多肽PrP106-126处理后,与转染空白载体的对照组相比,caspase-3剪切体和PARP剪切体的量明显下降。上述结果表明,过表达PRAS40可以缓解神经毒性多肽引起的神经元凋亡。(3)在神经毒性多肽PrP106-126处理后,与转染空白载体的对照组相比,转染了 HA-PRAS40的细胞表现出了 S6K1和4EBP1磷酸化水平的下降。mTOR信号通路在神经毒性多肽PrP106-126作用后可以被过度活化,而这种活化可以通过PRAS40的过表达或者雷帕霉素的处理而被抑制。(4)在过表达PRAS40之后,Akt和GSK3 β的磷酸化水平明显升高,表明PI3K-Akt信号通路被激活。加入Akt的抑制剂渥漫青霉素后,发现PRAS40对PrP106-126引起的神经元凋亡的抑制作用被减弱了,因此,PRAS40缓解PrP106-126引起的神经元凋亡,是通过恢复Akt的磷酸化活性来实现的。Akt可能部分参与了 mTOR调节的PrP106-126引起的神经元凋亡。PRAS40抑制了 mTORC1的过度活化,并且在保护细胞应对神经毒性多肽引起的凋亡中发挥了重要作用。因而,PRAS40作为靶蛋白,可为Prion疾病的治疗提供潜在的方法和思路。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S855.3

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