核酸对铜锌超氧化物歧化酶聚集的调控作用

发布时间：2018-07-11 10:12

本文选题：肌萎缩侧索硬化症 + SOD1　；参考：《华中师范大学》2014年硕士论文

【摘要】：肌萎缩侧索硬化症(Amyotrophic Lateral Sclerosis, ALS)是神经退行性疾病之一,研究发现它与错误折叠的SOD1聚集有关,其聚集形成的寡聚体和可溶低聚物被推测为细胞毒性物种。SOD1自身的或由外界环境引起的结构和性质的改变可能是导致SOD1聚集的主要原因,结合本课题组研究基础,本论文研究了不同序列的DNA与wtSOD1相互作用和聚集过程,促进SOD1形成不同形貌的聚集体,并探讨了SOD1形成的聚集体类型与细胞毒性之间的关系。本文主要通过等温量热滴定法、圆二色谱法、ANS荧光光谱、紫外吸收光谱、直角光散射法、倒置荧光显微镜、透射电镜及MTT法细胞毒性等实验,研究DNA与SOD1相互作用,取得结果如下： (1)等温量热滴定实验显示,各种序列的DNA与SOD1相互作用都是焓驱动的放热过程,主要通过静电相互作用结合,并且熵变不同。 (2)通过圆二色光谱实验可见,不同的DNA与SOD1相互作用,DNA二级结构不发生改变。ANS荧光实验表明,加入DNA使SOD1疏水残基进一步暴露,并随DNA浓度增加呈正相关,不同DNA使SOD1疏水残基暴露程度分为两组,Poly(dC)12、Poly(dG)12明显大于Poly(dA)12、Poly(dT)12,四链体[P1]、[P2]、[d(TG4T)]4明显大于相对应的ss DNA,特征峰出现了蓝移,说明加入DNA使SOD1构象进一步变化。 (3)直角光散射实验发现,不同DNA促进SOD1聚集反应快速达到平衡,形成的聚集体粒径大小也不同,分列两组,SOD1与Poly(dC)12、Poly(dG)12聚集体粒径明显大于Poly(dA)12、Poly(dT)12,当浓度增加,聚集体粒径随之增大。 (4)倒置荧光显微镜实验可见,Poly(dA)12、Poly(dT)12促使SOD1形成的聚集体荧光强度要弱于Poly(dG)12形成的聚集体。透射电镜法可看到纳米级的聚集体形貌,前者主要形成了无定形聚集体,后者主要形成了纤维状的聚集体。而CR紫外吸收实验也表明,四链体[P1]、[P2]、[d(TG4T)]4促使SOD1形成的聚集体淀粉样纤维结构要多于对应的ss DNA。 (5)MTT法细胞毒性实验发现,加入Poly(dA)12、Poly(dT)12、ss[P1]、ss[P2]、 ss[d(TG4T)]4形成的无定形聚集体培养的细胞成活率明显低于加入Poly(dC)12、 Poly(dG)12、四链体[P1]、[P2]、[d(TG4T)]4形成的纤维状聚集体,从实验数据探讨聚集体类型与细胞毒性之间的关系。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is one of the neurodegenerative diseases. The oligomers and soluble oligomers formed by SOD1 are presumed to be cytotoxic species. The changes in structure and properties caused by the external environment may be the main causes of SOD1 aggregation. In this paper, we studied the interaction and aggregation process between DNA and wtSOD1 of different sequences, promoted the formation of SOD1 aggregates with different morphologies, and discussed the relationship between the types of aggregates formed by SOD1 and cytotoxicity. The interaction between DNA and SOD1 was studied by means of isothermal calorimetric titration, circular dichroism ans fluorescence spectrum, UV absorption spectrum, right angle light scattering method, inverted fluorescence microscope, transmission electron microscope and MTT assay. The results are as follows: (1) the results of isothermal calorimetric titration show that the interaction between DNA and SOD1 is an enthalpy driven exothermic process, which is mainly combined by electrostatic interaction. And the entropy change is different. (2) through circular dichroism spectrum experiment, it can be seen that the secondary structure of DNA in different DNA interacting with SOD1 does not change. Ans fluorescence experiment shows that the addition of DNA further exposes the hydrophobic residue of SOD1. With the increase of DNA concentration, there was a positive correlation between the exposure degree of SOD1 hydrophobic residues in two groups: Poly (DC) 12N Poly (dG) 12 was significantly larger than Poly (dA) 12 Poly (DT) 12, the quadruplex [P1], [P2], [d (TG4T)] 4 were obviously larger than the corresponding ss DNAs, and the characteristic peaks showed blue shift. The results showed that the conformation of SOD1 was further changed by adding DNA. (3) the right-angle light scattering experiments showed that different DNA promoted SOD1 aggregation reaction to a rapid equilibrium, and the particle size of the aggregates formed was also different. The particle size of SOD1 and Poly (dG) 12 aggregates was obviously larger than that of Poly (dA) 12 Poly (DT) 12 groups, and the particle size increased with increasing concentration. (4) the fluorescence intensity of Poly (dA) 12 / Poly (DT) 12 was lower than that of Poly (dG) 12 aggregates induced by Pol (dG) 12. (4) the fluorescence intensity of poly (dA) 12 / Poly (DT) 12 was lower than that of Poly (dG) 12. The fluorescence intensity of the aggregates induced by poly (dG) 12 was lower than that of Poly (dG) 12. The morphology of nano-scale aggregates can be seen by transmission electron microscopy. The former mainly forms amorphous aggregates and the latter mainly forms fibrous aggregates. Cr UV absorption assay also showed that tetrastrons [P1], [P2], [d (TG4T)] 4 promoted SOD1 to form more amyloid fibers than ss DNA. (5) MTT assay showed that, The cell survival rate of amorphous aggregates formed by adding Poly (dA) 12 (DT) Poly (DT) 12n ss [P1] Pnss [P2], ss [d (TG4T)] 4 was significantly lower than that of fibrous aggregates formed by adding Poly (DC) 12, Poly (dG) 12, Four-chain bodies [P1], [P2], [d (TG4T)] 4. The relationship between the type of aggregates and cytotoxicity was discussed from the experimental data.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.8

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