【作者】 张建荣；

【导师】 刘文广；

【作者基本信息】 天津大学 ， 生物医学工程， 2004， 硕士

【摘要】 本文以 5－甲醛基－2－呋喃磺酸钠盐（FFSA）和精氨酸分别修饰壳聚糖，制备两种抗凝血生物医用材料。主要内容包括以下两部分：一、采用 5－甲醛基－2－呋喃磺酸钠盐（FFSA）和壳聚糖间的 Schiff 碱反应，制备出一系列不同分子量、不同取代度的 N－磺酸糠基壳聚糖。以红外光谱（FTIR）和元素分析对制得的 N－磺酸糠基壳聚糖进行表征。用圆二色谱仪（CD）测定壳聚糖、N－磺酸糠基壳聚糖对凝血酶构象的构象影响。结果表明随着 FFSA 加入量的增加，磺酸基在壳聚糖上的含量提高。凝血酶与 N－磺酸糠基壳聚糖作用较强，构象发生了很大的改变，而与壳聚糖间的作用很弱。原子力显微镜（AFM）形貌分析揭示，N－磺酸糠基壳聚糖表面能有效地阻止纤维蛋白原交联形成网状结构。测定活化部分凝血活酶时间（APTT）、血浆凝血酶原时间（PT）和凝血酶凝结时间（TT）来评价氧化葡萄糖醛交联的壳聚糖和 N－磺酸糠基壳聚糖材料表面抗凝血性能。结果表明，与正常人血浆对照组相比，不同分子量、不同取代度的 N－磺酸糠基壳聚糖的 APTT均有 10 秒以上的延长，并且延长的时间随 5－甲醛基－2－呋喃磺酸钠盐的取代度的增大而增大，但 PT 和 TT 没有病理意义上的延长，说明 N－磺酸糠基壳聚糖是通过内源性途径起抗凝血作用。但在同一取代度下，不同分子量N－磺酸糠基壳聚糖衍生物的 APTT 无明显差异。血小板的粘附结果表明，N－磺酸糠基壳聚糖膜材在一定的程度上能够阻止血小板的粘附与变形。细胞毒实验证明，在氧化葡萄糖醛含量低于 10％时，交联壳聚糖膜无细胞毒。二、用碳二亚胺偶联法制备了壳聚糖－精氨酸缀合物，FTIR、核磁共振、光电子能谱结果显示精氨酸键合到壳聚糖中。CD 谱揭示壳聚糖－精氨酸缀合物同样与凝血酶间存在强相互作用。APTT，PT 和 TT 的测定结果表明，葡萄糖醛交联壳聚糖－精氨酸缀合物膜材的 APTT 和 PT 值均有病理意义上的延长，APTT最长达71.2s,PT则达到21.6s,这说明壳聚糖－精氨酸缀合物是通过内源性和外源性两种途径共同起抗凝血作用的。并且 APTT 和 PT 的延长的程度会随着精氨酸加入量的增加而增大，但在同一加入量下，不同分子量壳聚糖的精氨酸衍生物的 APTT，PT 无明显差异。与 N－磺酸糠基壳聚糖相比，壳聚糖－精氨酸缀合物的抗凝血性更突出。更多还原

【Abstract】 In this work, two different anticoagulant biomaterials were prepared bymodifying chitosan with 5-formyl-2-furansulfonic acid (FFSA) and arginine,respectively. The main contents include two parts as follows:1. A series of N-sulfofurfuryl chitosans(N-Su-CSs) with different molecular weightsand substitution degrees were prepared by coupling FFSA to chitosan via Schiff’sbase reaction in the aqueous solution of acetic acid. The obtained N-Su-CSs werecharacterized by FTIR and elemental analysis. The change in the conformation ofthrombin caused by chitosan and N-Su-CS was evaluated by Circular Dichroism(CD).The result indicated that the contents of sulfonic acid in N-Su-CSs were raised withincreasing the addition amount of FFSA. The CD demonstrated that the N-Su-CSssignificantly altered the conformation of thrombin, whereas no obvious variation inthe conformation of thrombin was observed with the addition of chitosan. AFM imageshowed that the N-Su-CSs could prevent individual fibrinogen molecules fromaggregating and forming networks. The anticoagulation activity of oxidized glucose aldehyde-crosslinked N-Su-CSand chitosan membranes was examined by assaying activated partial thromboplastintime (APTT), prothrombin time (PT) and thrombin time (TT). The result indicatedthat APTTs of N-Su-CS membrane with different molecules and substitution degreeswere prolonged over 10s compared with that of the plasma of healthy people, andshowed a rising trend upon increasing the content of sulfonic acid groups. However,the PT and TT of modified chitosan remained the same level as that of chitosan,suggesting N-Su-CSs prevented blood coagulation via an intrinsic pathway. However,with the same substitution degree, the N-Su-CSs with different molecules showed nodistinct difference of APTT. The result of platelet adhesion on the N-Su-CS andchitosan membranes revealed that N-Su-CS could prevent the adhesion and activationof platelet to some extent. The preliminary experimental results of cytotoxicity ofoxidized glucose-crosslinked chitosan membranes demonstrated that the membraneswere non-cytotoxic with the content of crosslinker below10%.2. Chitosan was modified with arginine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. FTIR, C 13NMR and XPS spectra showed that arginine was chemically coupled to chitosan toform a chitosan-arginine conjugate (CS-ArgC). The CD spectra implied a strong<WP=5>interaction between CS-ArgC and thrombin. The results of APTT, PT and TT showedthat APTT and PT of glucose aldehyde-crosslinked CS-ArgC membranes wereprolonged with clinical significance compared with that of chitosan counterpart. TheAPTT reached a maximum value 71.2s, and PT 21.6s, indicating that CS-ArgChindered blood coagulation via both intrinsic and extrinsic pathway. Moreover, theincreasing extent of APTT and PT was dependent on the addition amount of arginine.But with the same substitution degree, the CS-ArgC with different molecules showedno obvious difference of APTT and PT. Compared with N-Su-CS, CS-ArgC was morenotable as an anticoagulant.更多还原