【作者】 刘锐；

【导师】 何志敏；

【作者基本信息】 天津大学 ， 化学工程， 2010， 硕士

【摘要】 病理学研究表明,至少20种淀粉样蛋白与神经变性疾病、糖尿病等顽疾密切相关,如阿尔茨海默氏病、II型糖尿病、帕金森综合症以及亨廷顿舞蹈症等。此外,因蛋白质变性而形成的聚集体,将给蛋白质药物的生产、贮藏与运输带来极大困难,同时对蛋白质药物的安全使用带来风险,是生物医药领域中亟待解决的技术问题。本文以模式药物蛋白胰岛素为研究对象,结合现代生物物理检测手段,对胰岛素聚集机理进行验证,建立新型淀粉样纤维体表征检测技术,提出氨水-过氧化氢两步法途径聚集淀粉样纤维和再生胰岛素二硫键,并开展了胰岛素低聚体和淀粉样纤维的相关抑制工作。具体研究内容和结论如下:第一,综合利用圆二色谱、荧光光谱、动态光散射、电子显微镜和太赫兹时域光谱多种现代检测技术,多尺度表征胰岛素淀粉样纤维化过程中二级、三级、四级结构的转变与状态密度的改变,验证了胰岛素聚集机理;首次利用太赫兹时域光谱仪研究和检测蛋白质淀粉样纤维化行为,获得0.2 THz².0 THz频率区的太赫兹吸收谱和折光系数变化曲线,建立了一种利用太赫兹时域光谱技术对淀粉样蛋白质进行快速、无损的检测方法。第二,尝试使用NH₃-H₂O₂两步法对胰岛素淀粉样纤维进行解聚与单体二硫键断裂和恢复,并对比NH₃-H₂O₂耦合法,确定了还原-氧化过程的关键因素;提出氨水对胰岛素淀粉样纤维解聚的机理模型,指出氨水作用机制为通过改变蛋白周围亲疏水环境以及破坏固有氢键作用,逐步释放单个胰岛素分子。第三,对比不同类型多羟基化合物对胰岛素聚集与淀粉样纤维化过程的影响,分别解释了原花青素、白藜芦醇和海藻糖加入20% HAc溶液和20% HAc-0.1 M NaCl溶液体系后,胰岛素聚集荧光动力学和原纤/纤维的结构形貌改变;指出抑制能力顺序:原花青素>白藜芦醇>海藻糖,分别起到抑制成核、阻碍生长和无明显抑制的作用。第四,采用532 nm波长的激光,通过抑制胰岛素成核机理,阻碍成熟纤维形成;特别指出在使用动态光散射仪和ThT荧光光谱仪对样品进行在线测量时,应注意所测样品是否易于发生光化学反应,如发生聚集和解聚。本实验亦证明了连续激光照射将为胰岛素提供一个相对稳定的溶液环境,有利于维持胰岛素低聚体状态,关于自由基作用机理方面具有一定的研究前景。更多还原

【Abstract】 At least 20 amyloid proteins are implicted in the pathologies of various human diseases such as Alzheimer’s, type II diabetes, Parkinson’s and Huntingtons’s. The denatured proteins forms aggregates which cause problems to the production, storage and transport of protein drugs, are quite dangerous to drug intaken. Therefore, it is critical to develop related inhibitors as well as to improve detection techniques. Our studies have verified the mechanism of insulin aggregation, and we first established THz TDS method used for detecting insulin amyloid fibrillation. In addition, we destructed the fibrils and regenerated the disulfide bonds of insulin by NH₃-H₂O₂ two-step method, and investigated the inhibition effects on insulin aggregation by small polyphenolic molecules and laser irradiation. The main ideas and conclusions are summarized as follows.Firstly, to study the changes in secondary, tertiary, and quaternary structures and the alteration in the collective vibrational mode density of states during the amyloid fibrillation, bovine insulin in 20% acetic acid was incubated at 60 oC, and its multi-level structures were followed by various biophysical techniques including CD, ThT, DLS, EM and THz absorption spectroscopy. There is an apparent increase in both the absorbance and refractive index in THz spectra, thus THz technique is expected to provide a quick and non-invasive way of looking into amyloid fibrillation. Secondly, the NH₃-H₂O₂ two-step method was investigated to regenerate insulin monomers and recover the structure. The key parameters were identified via comparision with NH₃-H₂O₂ coupling methond, and the model of insulin fibrils disaggregation by adding aqueous ammonia was supposed to disrupting the hydrophobic environment and intrinsic hydrogen bonds.Thirdly, the polyphenol was studied to inhibit insulin amyloid fibrillation. The inhibition ability of procyanidine, resveratrol and trehalose decreased in turn. They may effect the process of insulin fibrillation by inhibiting insulin nucleation, preventing growth of protofilament and with no significant inhibition effects, respectively.Fourthly, a fibrillation inhibition experiment using laser beam irradiation is introduced. Laser can prevent insulin from forming mature fibrils via nucleation inhibition. Importantly, it should be noted that online laser measurements such as dynamic light scattering and ThT fluorescence should be carefully monitored when samples are prone to aggregate or disaggregate. As shown in our results, continuous irradiation of the laser beam induced a comparable steady state of the unaggregated insulin, thereby preventing further fibrillation and, consequently, potentially curing related amyloid diseases.更多还原