HS类似物的化学酶法合成、生物活性及其对材料表面蛋白质吸附行为的影响

发布时间：2019-03-03 20:39

【摘要】：生物医用材料与血液相互接触时，首先血浆蛋白质会吸附到材料表面，从而触发后续的凝血级联过程，因此血浆蛋白质的吸附对后续的凝血过程起到决定性的作用。通过对材料表面改性以提高其阻抗血浆蛋白非特异性吸附能力，是提高生物医用材料血液相容性的有效方法之一。本文使用K5多糖合成具有不同硫酸化位点的硫酸乙酰肝素(Heparan sulfate，HS)类似物，探讨不同硫酸化位点的硫酸乙酰肝素类似物在材料表面与血浆蛋白质的吸附情况，以期得到一种硫酸乙酰肝素类似物，能够有效抵抗血浆蛋白质的非特异性吸附，用于构建蛋白质不吸附材料，改善材料表面的血液相容性。本文以LB为培养基，培养了大肠杆菌K5菌株，采用醇沉分离了K5粗多糖，对K5粗多糖进行了脱蛋白处理，采用DEAE阴离子交换树脂、G75葡聚糖凝胶对K5多糖进行了纯化，得到了糖醛酸含量为33.1%的K5多糖，并用红外、核磁对其进行了表征。采用三氧化硫吡啶法对K5多糖N位进行了硫酸化，成功制备了NSK5多糖，并用红外对其进行了表征。测定了其抗氧化活性，结果显示一定浓度范围内，NSK5多糖的抗氧化活性较K5多糖高。通过摇瓶培养成功表达了四种硫酸基转移酶AST-IV、2-OST、6-OST-1、3-OST-1。并用上述硫酸基转移酶对NSK5多糖进行了定位硫酸化修饰，合成了3种HS类似物，对合成的产物进行了二糖分析，测定了其抗Xa因子活性，结果显示硫酸基成功转移到特定的位点上，产物具有良好的抗Xa因子活性。将HS衍生物通过共价键接枝到硅片表面，并通过水接触角、XPS、AFM对接枝的情况进行了研究，结果显示HS衍生物成功接枝到硅片表面。最后采用125I同位素标记法研究了改性后硅片表面对纤维蛋白原的吸附情况，结果显示接枝了26NSK5和NSK5多糖的硅片，与氨基化处理后的硅片相比，对纤维蛋白原的吸附分别可以降低69.2%、90.5%。细胞粘附实验结果显示，63NSK5、26NSK5、263NSK5改性硅片对HMEC-1细胞的粘附效果均得到改善。
[Abstract]:When biomedical material and blood contact with each other, the plasma protein will first adsorb to the surface of the material, thus triggering the subsequent coagulation cascade process. Therefore, the adsorption of plasma protein plays a decisive role in the subsequent coagulation process. It is one of the effective methods to improve the blood compatibility of biomaterial by modifying the surface of the material to improve the nonspecific adsorption ability of impedance plasma protein. In this paper, acetylheparin sulfate (Heparan sulfate,HS) analogues with different sulfation sites were synthesized by using K5 polysaccharide, and the adsorption of heparin sulfate analogues with different sulfation sites on the surface of the materials was studied, and the adsorption of heparin sulfate analogues with plasma proteins was studied. In order to obtain a heparin sulfate analogue, it can effectively resist the non-specific adsorption of plasma protein, and can be used to construct protein-non-adsorptive material and improve the blood compatibility of the surface of the material. In this paper, Escherichia coli strain K5 was cultured on LB medium, the crude K5 polysaccharide was separated by alcohol precipitation, the crude K5 polysaccharide was deproteinized, the K5 polysaccharide was purified by DEAE anion exchange resin and G75 dextran gel. K _ 5 polysaccharide containing 33.1% uronic acid was obtained and characterized by IR and NMR. The N site of K5 polysaccharide was sulfated by thiopyridyl trioxide method. The NSK5 polysaccharide was successfully prepared and characterized by IR. The results showed that the antioxidant activity of NSK5 polysaccharide was higher than that of K5 polysaccharide in a certain concentration range. Four sulfate transferase AST-IV,2-OST,6-OST-1,3-OST-1. were successfully expressed in shaking flask culture. Three kinds of HS analogues were synthesized by sulfation modification of NSK5 polysaccharides with the above sulfate transferase. The synthesized products were analyzed by disaccharide analysis and their anti-Xa factor activities were determined. The results showed that the sulfate group was successfully transferred to a specific site, and the product had good anti-Xa factor activity. HS derivatives were grafted onto the surface of silicon wafers by covalent bonding, and the grafting conditions were studied by water contact angle and XPS,AFM. The results showed that the HS derivatives were successfully grafted onto the surface of silicon wafers. Finally, the adsorption of fibrinogen on the surface of modified silicon wafer was studied by 125i isotope labeling method. The results showed that the modified silicon wafer grafted with 26NSK5 and NSK5 polysaccharide was compared with the modified silicon wafer. The adsorption of fibrinogen decreased by 69.2% and 90.5% respectively. The results of cell adhesion test showed that the adhesion effect of 63 NSK5 and 26NSK5263NSK5 modified silicon wafers on HMEC-1 cells was improved.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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