以丝氨酸为配基的聚砜亲和膜及其内毒素脱除性能

发布时间：2018-03-10 07:14

本文选题：亲和膜　切入点：内毒素　出处：《浙江大学》2012年硕士论文　论文类型：学位论文

【摘要】：内毒素是革兰氏阴性菌细胞壁上的脂多糖成分,进入人体血液中后作用于免疫系统,产生强烈的生物学效应,普遍被认为与危重症疾病脓毒血症相关。亲和吸附法是清除血液中的内毒素最有效的方法之一,因此,开展亲和膜吸附内毒素的研究对促进脓毒血症的治疗具有非常重要的意义。本文以常见的血液透析膜材料PSF为基质膜材料,在保证膜材料主体性质不变的前提下,针对PSF耐有机溶剂性能差的特点,设计了一种可在常温下进行的PSF中空纤维膜表面改性方法：利用Friedel-Crafts反应向聚砜膜表面引入活性基团-CH2C1,以乙二胺为间隔臂,以戊二醛为活化剂,成功接枝配基L-Ser.膜上氨基酸密度可以通过控制第一步氯甲基化反应程度来控制,最适宜的氯甲基化时间为60min,此时膜上配基密度为2.04μmol/cm2。ATR-FTIR和XPS表征结果显示上述改性方法是有效的,静态接触角结果显示PSF-Ser亲和膜的亲水性与PSF膜相比有了显著提升。接枝Ser配基后,膜在去离子水、磷酸缓冲液和BSA溶液中对内毒素的特异性吸附均有所增强,其吸附性能受到溶液环境中pH、离子强度、内毒素浓度及蛋白质浓度等因素的影响。在与血液生理环境相似的磷酸缓冲液(pH7.0,0.15M)中,PSF-Ser亲和膜单位面积的内毒素吸附量为1.656EU/cm2。与实验室前期制备的PVDF-Ser亲和膜相比,PSF-Ser膜对内毒素的吸附能力增强了,对BSA的吸附差别不大。为了从分子层面讨论配基与内毒素作用机理,本文还建立了内毒素的活性保守结构——类脂A分子在CHARMM力场下的分子模型。其在水环境下的初态模拟构象结果为稳定的双分子层结构,当水质量含量为40%时,该双分子层的厚度为4.65nm/4.98nm,其结构与厚度均与文献报道一致,证明所用力场参数可靠。此外,对类脂A分子排列成的双分子层结构进行了原子级描述。上述研究结果表明,制备得到的PSF-Ser中空纤维亲和膜可特异性去除水溶液或蛋白质溶液中的内毒素,有望作为一种内毒素吸附膜应用在脓毒血症病人的血液净化治疗过程中,同时建立起的类脂A分子力场模型可用于后续分子动力学研究,为探讨内毒素与亲和配基之间的作用机理奠定基础。
[Abstract]:Endotoxin is a lipopolysaccharide component on the cell wall of Gram-negative bacteria. It acts on the immune system after entering the human blood and has a strong biological effect. Affinity adsorption is one of the most effective methods to remove endotoxin from blood. The study of adsorption of endotoxin by affinity membrane is of great significance in promoting the treatment of sepsis. In this paper, the common hemodialysis membrane material, PSF, is used as the matrix membrane material. On the premise of keeping the main properties of the membrane material unchanged, the characteristics of PSF's poor resistance to organic solvents are pointed out. A method of surface modification of PSF hollow fiber membrane at room temperature was designed. The active group -CH2C1 was introduced into the surface of polysulfone membrane by Friedel-Crafts reaction, with ethylenediamine as spacer and glutaraldehyde as activator. The densities of amino acids on the membrane can be controlled by controlling the degree of the first step chloromethylation reaction, and the optimum chloromethylation time is 60 minutes. The results of characterization of ligand density of 2.04 渭 mol/cm2.ATR-FTIR and XPS show that the above modification method is effective. The static contact angle showed that the hydrophilicity of PSF-Ser affinity membrane was significantly higher than that of PSF membrane. After grafting with Ser ligand, the specific adsorption of endotoxin in deionized water, phosphoric acid buffer and BSA solution was enhanced. Effects of endotoxin concentration and protein concentration on endotoxin adsorption of PSF-Ser affinity membrane in phosphate buffer (pH7.0 ~ 0.15M), which is similar to that of blood physiological environment. The endotoxin adsorption capacity per unit area of PSF-Ser affinity membrane is 1.656 EU. / cm ~ (2). Compared with the PVDF-Ser affinity membrane prepared in early laboratory, the PSF-Ser membrane is obtained. The ability to adsorb endotoxin increased, The adsorption of BSA has little difference. In order to discuss the mechanism of ligands acting on endotoxin at the molecular level, The molecular model of lipopolysaccharide A molecule under CHARMM force field is also established. The initial state simulation conformation of lipopolysaccharide A molecule under water environment is a stable bilayer structure, when the mass content of water is 40%, The thickness of the bimolecular layer is 4.65nm / 4.98nm.The structure and thickness of the bimolecular layer are consistent with those reported in the literature, which proves that the force field parameters used are reliable. In addition, the structure of bilimolecular layer arranged by lipoid A molecule is described at atomic level. These results indicate that the PSF-Ser hollow fiber affinity membrane can specifically remove endotoxin from aqueous solution or protein solution, which is expected to be used as a endotoxin adsorption membrane in blood purification treatment of sepsis patients. At the same time, the molecular force field model of lipids A can be used in the further study of molecular dynamics, which lays a foundation for the study of the interaction mechanism between endotoxin and affinity ligands.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TQ028.8;R318.08

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