【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(dòng)物中廣泛存在的致死性神經(jīng)退行性疾病。根據(jù)“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(gòu)象轉(zhuǎn)變形成的致病型朊蛋白,這種構(gòu)象的朊蛋白可以誘導(dǎo)更多的正常朊蛋白發(fā)生構(gòu)象轉(zhuǎn)變,進(jìn)而引發(fā)神經(jīng)毒性和神經(jīng)退行性病變。 在朊蛋白發(fā)生構(gòu)象轉(zhuǎn)變的過程中,目前認(rèn)為有某些輔助因子參與、促進(jìn)了朊蛋白構(gòu)象轉(zhuǎn)變的進(jìn)行,脂質(zhì)就是一種有效的輔助因子。研究表明帶負(fù)電荷的脂質(zhì)可以和朊蛋白相互作用并誘導(dǎo)其發(fā)生構(gòu)象轉(zhuǎn)變,并形成具有PK抗性的構(gòu)象體。此外RNA作為一種長鏈陰離子復(fù)合結(jié)構(gòu)的物質(zhì),對(duì)朊蛋白構(gòu)象轉(zhuǎn)變也有加速的作用。 在本論文中,我們?cè)隗w外條件下用蛋白質(zhì)錯(cuò)誤折疊循環(huán)擴(kuò)增的技術(shù),在體系中加入了大腸桿菌表達(dá)的重組朊蛋白、帶有負(fù)電荷的脂質(zhì)POPG和小鼠肝臟提取的總RNA進(jìn)行朊蛋白的構(gòu)象轉(zhuǎn)變,從無到有地制備出了重組朊病毒。經(jīng)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(diǎn),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(nèi)注射的方式感染野生型CD-1小鼠,在5個(gè)月的潛伏期后小鼠開始出現(xiàn)朊蛋白疾病的相關(guān)癥狀。在病程末期處死小數(shù)進(jìn)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(cuò)誤折疊構(gòu)象,具有傳染性和致病性的特點(diǎn))、腦組織海綿狀空泡、異常朊蛋白聚集、膠質(zhì)細(xì)胞增生等典型的朊蛋白疾病病理學(xué)特點(diǎn),表明該重組朊病毒確實(shí)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(zhì)作為輔助因子以及RNA的幫助下,在體外用PMCA技術(shù)從無到有地制備出朊病毒,并且該朊病毒具有很強(qiáng)的傳染性和致病性,這就強(qiáng)有力的支持了“朊病毒”假說,并且為進(jìn)一步研究朊蛋白的構(gòu)象轉(zhuǎn)變提供了良好的實(shí)驗(yàn)平臺(tái)。
[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(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373;R-332

【共引文獻(xiàn)】

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1 Wei Wang;Xiao Li Yan;Lei Zhan;Fei Leng;Xiao Xi Yang;Cheng Zhi Huang;;Cellular prion protein imaging analysis with aptamer-labeled Ru(bpy)_3~(2+)-doped silica nanoparticles[J];Chinese Science Bulletin;2014年02期

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

3 Zhiqi Song;Deming Zhao;Lifeng Yang;;Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein[J];Neural Regeneration Research;2013年30期

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1 崔培昕;朊蛋白和去甲酰化酶結(jié)構(gòu)與性質(zhì)的X射線譜學(xué)研究[D];中國科學(xué)技術(shù)大學(xué);2013年

2 龐曉靜;BRET方法研究BST-2和HIV-1 Vpu相互作用及小分子化合物抑制劑的篩選[D];北京協(xié)和醫(yī)學(xué)院;2013年

3 劉少卿;不同棉花種質(zhì)資源耐熱性鑒定及熱激效應(yīng)分析[D];中國農(nóng)業(yè)科學(xué)院;2011年

4 刁小龍;雞朊蛋白參與DF-1細(xì)胞增殖粘附的功能研究[D];甘肅農(nóng)業(yè)大學(xué);2013年

中國碩士學(xué)位論文全文數(shù)據(jù)庫 前6條

1 朱淼;銅離子對(duì)朊蛋白轉(zhuǎn)基因線蟲的影響[D];黑龍江八一農(nóng)墾大學(xué);2013年

2 岳宗偉;兩種淀粉樣纖維蛋白的表達(dá)純化及結(jié)構(gòu)的初步表征[D];華東師范大學(xué);2013年

3 呂敏;同種異體富血小板血漿標(biāo)準(zhǔn)化制備的研究[D];第四軍醫(yī)大學(xué);2013年

4 吳星亮;干擾素應(yīng)答分子Tetherin對(duì)抗馬傳染性貧血病毒的作用機(jī)制研究[D];中國農(nóng)業(yè)科學(xué)院;2013年

5 劉章;細(xì)胞型朊蛋白在不同月齡小鼠視網(wǎng)膜中表達(dá)的實(shí)驗(yàn)研究[D];南昌大學(xué)醫(yī)學(xué)院;2013年

6 陸小亮;蛋白質(zhì)折疊理論研究[D];華東師范大學(xué);2013年

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本文編號(hào)：2180775