低密度脂蛋白亲和膜表面模型化构建与相互作用研究

【作者】 李晶；

【导师】 徐志康； 黄小军；

【作者基本信息】 浙江大学 ， 高分子化学与物理， 2013， 硕士

【摘要】 人体血液中低密度脂蛋白(LDL)含量过高会引发心脑血管疾病,严重威胁人类健康。对于重症高血脂症患者的治疗,低密度脂蛋白血液净化疗法因其高效性而长期备受关注。近年来,各种血液净化疗法被广泛应用于临床医学研究,其研发核心为低密度脂蛋白亲和吸附材料。本文旨在通过分子自组装法构建富含糖基／磺酸基的模型表面,系统研究LDL蛋白与模型表面之间的相互作用过程,重点关注糖基在相互作用过程中发挥的协同效应,并提出“多重相互作用模型”；在此基础上,组合紫外光辐射接枝技术与“点击”化学表面改性方法,制备低密度脂蛋白亲和改性聚砜膜,用于LDL高效亲和吸附分离过程。具体研究内容如下：首先,通过混合自组装单分子层(mixed-SAM)法构建不同糖基/磺酸基组成的模型表面,利用表面等离子体共振(SPR)技术研究固-液体系低密度脂蛋白吸附动力学；实验结果显示,随着自组装模型表面糖基含量的增加,表面吸附蛋白量在糖基／磺酸基摩尔比为1.20时达到极大值,藉此提出“多重相互作用模型”,即除了被普遍认可的磺酸基与低密度脂蛋白间静电相互作用外,表面固定的糖基也参与了蛋白相互作用过程,表现为蛋白吸附过程中的协同效应；采用圆二色谱(CD)技术进一步研究糖基与对蛋白三维构象的影响,结果表明糖基促使LDL蛋白三级结构发生解螺旋现象。基于上述“多重相互作用模型”,通过紫外光辐射法引发丙烯酸(AA)在聚砜膜表面接枝聚合,再以EDC/NHS介导酰胺化反应与巯基糖/巯基磺酸类巯基试剂的巯-炔点击反应实现聚砜膜表面糖基/磺酸基化改性,制备LDL高效亲和吸附改性聚砜膜；采用酶联免疫吸附测试(ELISA)法系统研究改性膜表面蛋白吸附与脱吸附行为,当改性聚砜膜表面糖基/磺酸基摩尔比为0.95时,其对LDL蛋白吸附量出现极大值0.49μg/cm2,洗脱剂NaCl/Urea(1:1)溶液对糖基/磺酸基混合组成表面脱吸附效率高于单一羧基、磺酸基、糖基组成表面；上述实验结果在糖基/磺酸基化改性聚砜膜复杂形貌表面进一步验证了“多重相互作用模型”的适用性。本论文首创性提出的“多重相互作用模型”有望为新型低密度脂蛋白亲和吸附材料的研制开辟新思路。更多还原

【Abstract】 Abnormally high level of plasma low-density lipoprotein (LDL) in human plasma is a key pathogenic factor that contributes to atherosclerosis and finally leads to coronary artery diseases (CAD). LDL-ahperesis has been of greatest concern for its high efficiency in removing LDL, especially during the treatment of patients with severeyperlipidemia. For over25years, a variety of LDL-reduction therapies have been established for clinical application, and a majority of researches have been concentrated on the adsorbents with excellent affinity to LDL. Therefore, the thesis is focused on the adsorption/desorption processes between LDL protein and surfaces modified with glycosyl and sulfonic groups to achieve an extremely high affinity to LDL. The main contents are listed below:Model surfaces were modified with different ratios of glycosyl to sulfonic group via mixed self-assembled monolayer (SAM) method, and the kinetics of the LDL adsorption processes with those mixed-SAMs were studied by surface plasmon resonance (SPR) technique. Results showed that the adsorbed amount of LDL on different modified model surfaces changed with the ratio of glycosyl to sulfonic groups, and had a maximum when it reached1.20. Therefore, the ’Multiple-Interaction Model’ was suggested to describe the synergism effect of the glycosyl group during the LDL adsorption process, which inferred the other interactions between glycosyl groups and LDL protein, beyond the electrostatistic interaction that had long been acquainted between sulfonic groups and LDL. Circular dichroism (CD) spectrum further affirmed the conclusion above by the detection of tertiary structure transformations of LDL protein when substances with glycosyl groups were added into.Therefore, according to the’ Multiple-Interaction Model’, the polysulfone (PSf) membranes were firstly UV irradiated and grafted with acrylic acid, then followed by the amidation and ’thiol-yne’ click modification to realize a glycosylation and sulfonation process, and finally to achieve the high affinity to LDL protein. The enzyme-linked immunosorbent assay (ELISA) was applied to investigate the adsorption and desorption process of those modified PSf membranes. Results suggested a maximum adsorbed amount of LDL as high as0.49μg/cm2, when the ratio of glycosyl to sulfonic groups of modified PSf membranes was found to be0.95. Meanwhile, the desorption efficiencies using NaC1/Urea (1:1) solution, of those membranes with both glycosyl and sulfonic groups, were higher than ones with single functional groups. All evidence above strongly supports that the ’Multiple-Interaction Model’can also be used for the membrane surfaces with complicated morphologies.This first-presented’Multiple-Interaction Model’should be helpful to the design and fabrication of new materials with high LDL affinity.更多还原