化合物3d与HSA相互作用研究及MCF7细胞适配体筛选

发布时间：2018-03-18 06:42

本文选题：人血清白蛋白　切入点：黄酮衍生物　出处：《清华大学》2014年硕士论文　论文类型：学位论文

【摘要】：人血清白蛋白（HSA）是一个研究药物与蛋白质相互作用的模式蛋白。在本研究中，我们对实验室设计合成的化合物3d（对肝癌类细胞HepG2有潜在抗增值活性）与HSA的相互作用进行了探索。研究中使用荧光光谱法、紫外-可见光光谱法、圆二色谱法和分子模拟手段对二者相互作用情况进行表征。荧光光谱法实验结果表明HSA的荧光发射可以被3d明显的淬灭，并且最大发射波长从360nm红移到了363nm。利用Stern-volmere方程和修饰的Stern-volmere方程计算可知3d对HSA的荧光淬灭属于静态淬灭，二者在298K时的结合常数为5.26±0.04×104L mol-1。经过计算相关热力学参数得知，静电作用力在3d-HSA复合物稳定中起到了主要作用。从紫外-可见光光谱和圆二色谱结果都可以看出，HSA在与3d结合之后构象发生了改变，而且随着3d浓度的加大HSA结构变得松散、α-螺旋含量略有降低。分子模拟结果显示化合物3d可以恰好进入HSA的一个亚结构域IIA，而这个区域与HSA的储存和转运功能相关。该区域周边氨基酸残基ARG218、ARG222和LYS199与3d相应基团形成了三个正离子-π相互作用以及三个氢键作用。总之，黄酮类衍生物3d可以与HSA以非共价键方式中等结合力相结合，这样它就可以被HSA在血液中进行转运。另外还利用SELEX技术结合RFD分子模型对乳腺癌细胞MCF7进行了适配体的筛选工作。研究中使用MCF7细胞的培养液作为筛选的对象，根据细胞外分泌物的不同区分不同种类的癌细胞。通过24轮重复筛选和富集，最终得到了与MCF7细胞外液反应的分子库。克隆和测序之后获得了一些确定寡核苷酸序列，对其中的某些序列研究显示，它们均可以与MCF7的外液进行反应，获得明显的荧光信号。对它们细胞识别特异性研究发现不仅可以识别MCF7细胞，还对两种肝源的细胞HepG2和HL7702有较强的反应。另外对MCF10A的反应信号也较强，说明在这几种细胞的分泌物中都具有某一种或一类分子，，而这些分子恰好被筛选到的适配体分子库所识别。对序列ABE2-1细胞灵敏度检测实验表明，其最低检测细胞浓度可以达到103细胞/mL。采用超滤膜对细胞外液进行分段的方式初步确定靶标的分子量范围,最终确定靶标分子量范围在30-50kD之间。
[Abstract]:Human Serum Albumin (HSA) is a model protein that studies the interaction between drugs and proteins. We have explored the interaction of compounds designed and synthesized in laboratory for 3 d (with potential antiproliferative activity to hepatoma cell HepG2) with HSA. The fluorescence spectroscopy, UV-Vis spectroscopy, UV-Vis spectroscopy were used in the study. The interaction between the two was characterized by circular dichroism and molecular simulation. The fluorescence spectra showed that the fluorescence emission of HSA could be quenched by 3 d. The maximum emission wavelength was shifted from 360 nm to 363nm.The fluorescence quenching of HSA at 298K was statically quenched by Stern-volmere equation and modified Stern-volmere equation. The binding constant was 5.26 卤0.04 脳 10 4 L mol -1. Electrostatic force plays an important role in the stabilization of 3d-HSA complex. The conformation of HSA has changed after 3 d binding from UV-Vis spectra and circular dichroism spectra. Moreover, with the increase of 3D concentration of HSA, the content of 伪 -helix decreases slightly. The molecular simulation results show that the compound 3D can enter a subdomain of HSA, which is related to the storage and transport function of HSA. The amino acid residues ARG218N ARG222 and LYS199 formed three positive ion-蟺 interactions and three hydrogen bonds with their 3D corresponding groups. The flavonoid derivatives can bind to HSA in a non-covalent bond form for 3 d, so that it can be transported by HSA in the blood. In addition, the aptamer of breast cancer cell MCF7 was screened by SELEX technique and RFD molecular model. The culture medium of MCF7 cells was used as the screening object. According to different extracellular secretions, different types of cancer cells were differentiated. After 24 rounds of repeated screening and enrichment, a molecular library of extracellular fluid reaction with MCF7 was obtained. After cloning and sequencing, some definite oligonucleotide sequences were obtained. Studies on some of the sequences showed that they could react with the external fluid of MCF7 to obtain obvious fluorescence signals. The recognition specificity of their cells was found to recognize not only MCF7 cells, but also MCF7 cells. There was also a strong response to HepG2 and HL7702 in two liver-derived cells, and a stronger signal to MCF10A, indicating that there was one or a class of molecules in the secretions of these cells. These molecules were identified by a library of aptamer molecules that were screened. Sensitivity tests for sequenced ABE2-1 cells showed that, The lowest cell concentration can reach 103 cell / mL. The molecular weight range of target is determined by using ultrafiltration membrane to segment the extracellular fluid, and the molecular weight range of target is between 30 and 50 KD.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96;O657.3

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