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目的体外评价壳聚糖/肝素(Chi/Hep)共价包被先天性心脏病介入封堵材料的生物相容性。方法在南京医科大学第一附属医院心血管内科于2005年11月至2006年10月,通过体外实验检测Chi/Hep共价包被镍钛合金片的血液相容性和细胞相容性。实验分成血液对照组、未包被组、基础包被组、Chi/Hep包被组,其中按肝素质量浓度又分为低(0.1mg.mL-1)、中(1mg.mL-1)、高(10mg.mL-1)3个剂量组。Chi/Hep共价交联于试验用镍钛金属表面,(1)血液相容性实验:通过血液溶血试验,动态人全血接触试验评价其红细胞毒性及抗栓性能,扫描电子显微镜观察镍钛金属表面血小板粘附、聚集。(2)细胞相容性:利用纤维连接蛋白、Ki67免疫荧光标记人脐静脉内皮细胞(HUVECs)观察其细胞毒性。结果(1)血液相容性实验:实验各组红细胞、白细胞、血小板计数均在正常范围内,且组间比较差异无显著性意义,各实验组溶血率均小于5%。Chi/Hep包被组部分凝血活酶活化时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)均明显延长,血液对照组、未包被组及基础包被组两两比较差异无显著性意义,不同质量浓度肝素包被之间差异亦无显著性意义(APTT、PT方差均齐LSD、SNK法检验,TT方差不齐Dunnetts法检验P<0.05)。扫描电镜结果显示,Chi/Hep包被组金属表面少量血小板粘附。(2)细胞相容性实验:经过与HUVECs72h孵育,金属片边缘细胞生长、移行良好,无细胞变形;Fn免疫荧光标记HUVECs,发现不同包被镍钛金属片表面的HUVECs粘附顺序如下:基础包被组>未包被组>Chi/Hep组;Ki67免疫荧光标记HUVECs,随机双盲法计数细胞发现不同包被镍钛金属片表面的HUVECs增殖顺序如下:基础包被组=未包被组>Chi/Hep组(P<0.01)。结论体外观察Chi/Hep共价包被镍钛金属有良好抗血栓作用,但抑制内皮细胞粘附、增殖。
Objective To evaluate the biocompatibility of chitosan / heparin covalently implanted in congenital heart disease interventional blocking materials. Methods The blood compatibility and cytocompatibility of Chi / Hep covalently coated NiTi plates were tested in vitro from November 2005 to October 2006 in Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University. The experiment was divided into the blood control group, the uncoated group, the basic coated group and the Chi / Hep coated group. The heparinized group was divided into low (0.1mg.mL-1), medium (1mg.mL-1) High (10mg.mL-1) 3 dose groups. Chi / Hep was covalently cross-linked to the NiTi metal surface for testing. (1) Hemocompatibility test: The erythrocyte toxicity and antithrombotic properties were evaluated by hemolysis test and dynamic human whole blood exposure test. The NiTi Metal surface platelet adhesion, aggregation. (2) Cytocompatibility: Cytotoxicity was observed by using fibronectin and Ki67 immunofluorescence labeling of human umbilical vein endothelial cells (HUVECs). Results (1) Hematocompatibility test: The erythrocytes, white blood cells and platelet count in each group were within the normal range, and there was no significant difference between the two groups. The hemolysis rates of all experimental groups were less than 5%. The partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) in Chi / Hep group were significantly longer than those in the control group, non-coated group and basic coated group There was no significant difference between the two groups. There was no significant difference between different concentrations of heparin (APTT, PT, LSD, SNK, Dunnetts test, P <0.05). Scanning electron microscopy showed that a small amount of platelets adhered to the metal surface of Chi / Hep group. (2) Cytocompatibility experiment: After incubated with HUVECs for72h, the edge of the metal plate grew well and migrated well without cell deformation. Fn immunofluorescence labeled HUVECs showed that the order of HUVECs adhesion on the surface of NiTi plates was as follows: Coated group> uncoated group> Chi / Hep group; Ki67 immunofluorescence labeled HUVECs, random double-blind counting cells found that different coated nickel titanium metal surface HUVECs proliferation sequence is as follows: basic coating group = uncoated group > Chi / Hep group (P <0.01). Conclusions Chi / Hep covalently coated with NiTi has good antithrombotic effect in vitro, but inhibits the adhesion and proliferation of endothelial cells.