【摘要】：朊蛋白疾病,也被稱作傳染性海綿狀腦病,是一類在哺乳動物中廣泛存在的致死性神經退行性疾病。根據“朊病毒”假說,朊蛋白疾病的致病因子是朊病毒,其主要致病因子是由正常型的朊蛋白發(fā)生了構象轉變形成的致病型朊蛋白,這種構象的朊蛋白可以誘導更多的正常朊蛋白發(fā)生構象轉變,進而引發(fā)神經毒性和神經退行性病變。 在朊蛋白發(fā)生構象轉變的過程中,目前認為有某些輔助因子參與、促進了朊蛋白構象轉變的進行,脂質就是一種有效的輔助因子。研究表明帶負電荷的脂質可以和朊蛋白相互作用并誘導其發(fā)生構象轉變,并形成具有PK抗性的構象體。此外RNA作為一種長鏈陰離子復合結構的物質,對朊蛋白構象轉變也有加速的作用。 在本論文中,我們在體外條件下用蛋白質錯誤折疊循環(huán)擴增的技術,在體系中加入了大腸桿菌表達的重組朊蛋白、帶有負電荷的脂質POPG和小鼠肝臟提取的總RNA進行朊蛋白的構象轉變,從無到有地制備出了重組朊病毒。經鑒定該朊病毒具有聚集化、PK抗性且抗性肽段在C端的特點,與生理狀況下的朊病毒相似。 在體外制備出重組朊病毒之后,我們通過顱內注射的方式感染野生型CD-1小鼠,在5個月的潛伏期后小鼠開始出現朊蛋白疾病的相關癥狀。在病程末期處死小數進行生化和組化檢測,其具有腦部存在大量PrPsc(朊蛋白的一種錯誤折疊構象,具有傳染性和致病性的特點)、腦組織海綿狀空泡、異常朊蛋白聚集、膠質細胞增生等典型的朊蛋白疾病病理學特點,表明該重組朊病毒確實可以引發(fā)小鼠朊蛋白疾病。 總的來說,用重組朊蛋白可以在脂質作為輔助因子以及RNA的幫助下,在體外用PMCA技術從無到有地制備出朊病毒,并且該朊病毒具有很強的傳染性和致病性,這就強有力的支持了“朊病毒”假說,并且為進一步研究朊蛋白的構象轉變提供了良好的實驗平臺。
[Abstract]:Prion disease, also known as infectious spongiform encephalopathy, is a widespread fatal neurodegenerative disease in mammals. According to the prion hypothesis, prion disease is caused by prion, and its main pathogenic factor is the conformational transition from normal prion protein to pathogenetic prion protein. This conformational prion protein can induce more normal prion proteins to undergo conformational transformation, which leads to neurotoxicity and neurodegenerative lesions. In the process of conformation transformation of prion proteins, it is considered that there are some auxiliary factors involved in the conformation transition of prion proteins, and lipid is an effective auxiliary factor. It is shown that negatively charged lipids can interact with prion proteins and induce conformation transformation and form conformation bodies with competitive resistance. In addition, as a long chain anion compound structure, RNA also accelerates the conformation transition of prion protein. In this paper, we added the recombinant prion protein expressed by E. coli into the system by using the technique of protein misfolding cycle amplification in vitro. The recombinant prions were prepared by conformational transformation of prion protein with negative charge lipid POPG and total RNA extracted from mouse liver. It was identified that the prion had the characteristics of agglomeration PK resistance and the resistance peptide was at the C-terminal, which was similar to that of the prion in physiological condition. After the recombinant prion was prepared in vitro, we infected the wild type CD-1 mice by intracranial injection. After the incubation period of 5 months, the mice began to develop the related symptoms of prion disease. At the end of the course of the disease, the small number was killed for biochemical and histochemical examination. It had a large number of PrPsc in the brain (a misfolded conformation of prion protein, with infectious and pathogenicity), cavernous vacuole in brain tissue, abnormal aggregation of prion protein. The pathological features of some typical prion diseases such as glial cell proliferation indicate that the recombinant prion can indeed cause prion disease in mice. In general, the recombinant prion protein can be used to prepare prions from scratch in vitro with the help of lipids and RNA, and the prions are highly infectious and pathogenicity. This strongly supports the prion hypothesis and provides a good experimental platform for the further study of the conformation transition of prion proteins.
【學位授予單位】：華東師范大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R373;R-332

【共引文獻】

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2 Siqi Wang;Hui Zhao;Yaping Zhang;;Advances in research on Shadoo, shadow of prion protein[J];Chinese Science Bulletin;2014年09期

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