重组蛇毒纤溶因子rF Ⅱ偶联到聚氨酯表面及其纤溶活性评价

Immobilization of Recombinant Fibrinolytic Enzyme rF Ⅱ to Polyurethane Surface and Fibrinolytic Activity Evaluation

目的:通过聚氨酯表面偶联重组蛇毒纤溶因子rF Ⅱ来提高其纤溶活性。方法:在聚氨酯(PU)表面上涂覆含羧基硬段的聚氨酯溶液(PU1)制备羧基化表面聚氨酯(CPU),并用次甲基蓝吸附法测定表面羧基含量;在CPU表面上用EDC接枝双端氨基聚乙二醇(PEG),并用滴定法测定该表面的PEG接枝量;在CPU-PEG表面用EDC偶联重组蛇毒纤溶因子(rF Ⅱ),并用放射性同位素法测定该表面的rF Ⅱ偶联量,最后用试管定量法评价样品的纤溶活性。结果:CPU表面羧基含量为6.9μmol/cm2,CPU-PEG2k和CPU-PEG4k表面PEG含量分别为0.162和0.180μmol/cm2,CPU-PEG2k-125-ⅠrF Ⅱ和CPU-PEG4k1-25-ⅠrF Ⅱ表面rF Ⅱ含量分别为13.6和38.9ng/cm2,与对照样品相比都有很大的提高。纤溶活性评价表明,所有偶联rF Ⅱ样品的纤溶活性都有提高,抗血栓性能得到有效改善。结论:具有PEG间隔臂偶联rF Ⅱ的样品其纤溶活性比没有PEG间隔臂的样品更优,采用PEG4k间隔臂偶联rF Ⅱ样品的纤溶活性为最高。

Objective： Immobilizing rF Ⅱ, a recombinant fibrinolytic enzyme from agkistrodon acutus venom, to polyurethane to improve the surface antithrombogenicity. Methods： A solution of polyurethane with carboxylic groups in hard segment （ PU1 ） was coated on the substrate surface. The carboxylic group amount on the substrate surface was measured by methylene blue adsorption. PEG with an amino group at the molecular chain end was then covalently linked to the carboxylic group by EDC coupling. The amount of grafted PEG was determined by titration, rF Ⅱ was then immobilized to the amino groups of PEG by EDC coupling reaction. The amount of rF Ⅱ linked to the surfaces was measured by isotopes tagging. Results： The amount of carboxylic group on the substrate surface was 6.9μmol/ cm^2, the amount of PEG linked to the CPU-PEG2k or CPU-PEG4k surfaces was 0. 162 and 0. 180μmol/cm^2 respectively. The amount of rF Ⅱ immobilized to the CPU-PEG2k-^125 Ⅰ-rF Ⅱ or CPU PEG4k-^125 Ⅰ -rF Ⅱ surfaces was 13.6 and 38.9ng/cm^2 respectively. Evaluation results for the final resulting surface indicated that fibrinolytic activity of rF Ⅱ-immobilized surface was enhanced significantly compared with the controlled surface, and the antithrombogenicity was improved effectively as well. Conclusions ： Immobilization of rF Ⅱ with PEG spacer improved fibrinolytic activity of polyurethane surface.