【作者】 张东威；

【导师】 赵节绪；

【作者基本信息】 吉林大学 ， 神经病学， 2005， 博士

【摘要】 本研究通过建立朊蛋白106-126 肽段（PrP106-126）感染分化PC12细胞模型,利用流式细胞术、激光共聚焦显微镜技术、免疫组化以及蛋白质印记等技术从形态和功能两个方面对PrP106-126在分化PC12细胞上的毒性作用、具体的作用机制以及该方法能否作为朊蛋白病的发病模型进行了研究,观察了从早期出现的氧化应激到最终细胞凋亡的过程。朊蛋白病是一类由具有传染性的朊蛋白（Prion protein,PrP）为病原体所致的散发的人畜共患的中枢神经系统变性疾病,是一组既有传染性又有遗传性的变性疾病。朊蛋白疾病的核心是朊蛋白的构型由正常的PrPC向致病的PrPSC的转化,其共同的病理学特征是脑的海绵状变性。但是在发病机制上,PrPSC在脑内沉积如何引起继发的病理变化,目前尚不清楚,这也是朊蛋白病一直缺乏针对性治疗的原因之一。鉴于PrP106-126 具有许多PrPSC的生物学特性,通过建立PrP106-126 感染分化PC12 细胞模型,本研究发现,PrP106-126 对分化PC12 细胞具有确切的毒性作用,细胞感染PrP106-126 后较早出现氧化应激现象,并且氧化应激持续存在,随后出现细胞内钙离子浓度增加、线粒体膜电位的下降、Ca²⁺ATP 酶活性降低,细胞能量代谢受到严重干扰,最终导致细胞凋亡,Bcl-2/Bax 系统稳态的变化和Caspase-3 的活化参与了诱导凋亡的过程。本研究认为PrP106-126 感染分化PC12 细胞是研究朊蛋白病的高效模型,PrP106-126 的毒性作用是通过持续的氧化应激、干扰细胞的能量代谢、钙超载、最终诱导宿主细胞的凋亡来实现的,并且氧化应激处于始动与核心的地位,提示从抗氧化损伤角度着手筛选药物可能是在朊蛋白病治疗上的一种值得尝试的方法。更多还原

【Abstract】 It is believed that prion diseases are infectious, inherited, sporadic spongiform degeneration encephalopathies caused by prion protein conformational change. These disorders include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) of cattle, and Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS) and fatal familial insomnia (FFI) of humans. CJD is a rare disease that occurs in three forms: sporadic, genetic, and iatrogenic. More recently, a new variant of CJD (nvCJD) has been reported. All forms of transmissible spongiform encephalopathyes (TSEs) are characterized by spongiform degeneration of the brain, reactive gliosis, and neuronal loss. They are associated with the accumulation of an abnormal isoform (PrPsc) of the cellular prion protein (PrPc) in the brain. The scrapie prion protein, PrPsc, which is formed from PrPc, is assumed to be the etiological agent of TSE. In the transmissible particles, there is no evidence for a nucleic acid, which could be involved in the infection. Prion diseases have become an important issue not only in public health due to the possibility of plausible relationship between BSE and new variant CJD (nvCJD) but also in the scientific world due to the unique biological features of the prion agent. PrPc is a protein localized on the cell surface predominantly in brain, while PrPsc accumulates intracellularly in cytoplasmic vesicles. In contrast to PrPc, PrPsc has a high β-sheet content. Therefore, it is thought that this deleterious protein is formed by conversion of the putative α-helix of PrPc into β-sheets. No amino acid sequence or posttranslational differences have been detected between the normal host cell surface protein, PrPC, and its pathological form, PrPSc. The conversion of PrPC into PrPSc involves a conformational change whereby the α-helical content diminishes and the amount of β-sheet increases. The recognizable change between PrPC into PrPSc is acquisition of PI-PLC resistance, detergent insolubility and protease resistance. It has not been known how accumulation of PrPsc results in the subsequent changes, but there are evidences that it involves neuronal receptors, intracellular Ca2+ level and the balance of production of reactive oxygen species (ROS) and antioxidant defense mechanisms, and so on. Besides, apoptosis plays an important role in the etiology of prion disease. Apoptosis or programmed cell death is an important physiological process resulting in the controlled elimination of cells, e.g. during the development of the nervous system. In apoptosis nuclear chromatin undergoes condensation during which a calcium-dependent endonuclease is activated which cleaves nuclear DNA at linker regions, resulting in DNA fragments of 180-200bp in length and multiples of them. Apoptosis can be induced by a variety of stimuli, including heat shock, DNA damaging reagents and toxins, etc. Series of protease activation cascades involve in the apoptosis. Neuronal loss resulted from apoptosis is consistent with the absence of inflammatory react. PrP106-126, a synthetic peptide consisting of amino-acid residues 106-126 of human PrPc, is able to polymerize into amyloid-like fibrils in vitro, like PrPsc. It is neurotoxic and induces activation of astroglial and microglial cells in vitro. So PrP106-126 can serve as a model to study the cellular effects of PrPsc.In this experiment, the PC12 cells were infected by prion protein 106-126 peptide after differentiated by nerve growth factor (NGF). Cell viability or cell death was determined and cytotoxic effects and morphological changes were observed. The oxidative stress, energy metabolise and the calcium ion level were also detected. And the expression of Bcl-2 and Bax were detected by immunohistochemical technique. Flow cytometry and DNA electrophoresis were used to investigate whether apoptosis played a role in the pathogenesis. The activity of Caspase-3 was detected by western blot to study the function mode of apoptosis in the prion disease. The objective of this study is to evaluate the cytotoxic effects of prion protein 106-126 peptide, evaluate the possibility of the prion disease model which use differentiated PC12 cell infected by prion protein 106-126 peptide, and also evaluate the mechanism and effects of apoptosis in this model. Infected by this peptide, cell viability decreased along with the time the peptide infect and along with the dosage the peptide infect. Decrease of mitochondrial membrane potential were detected by flow cytometry, the activity of Ca2+ATPase decreased and increased reactive oxygen species (ROS) were detected by spectrophotometer ,and also the increased calcium ion in the cells were detected by confocal laser scanning microscopy. Under light microscope, cells were shrinked and rounded, many cells were divorced from plate wall, some neuraxon shortened and broken. Apoptosis cells which nucleolus shrinked and rounded could be coloured orange by fluorescent colouration. Under electron microscope, chromatin gathered along the inside of the nuclear membrane, vacuole bodies appeared. In the histogram of fluorescent staining of PI, a characteristic sub-G1 peak appeared before the diploid peak (G1/G0 phase) when treated with PrP106-126, apoptosis accounted for 28.2%, while the control group did not appear these sub-G1 peak. And also the ladder band appeared in DNA electrophoresis in the treat group. This phenomenon suggests apoptosis occurs in primary neuron culture, PrP106-126 induces neuronal apoptosis greatly. This might be a perfect model to study the cellular toxicity of prion protein.更多还原