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目的:体外评价壳聚糖/重组水蛭素(Chi/r-Hir)共价包被先天性心脏病介入封堵材料镍钛合金的生物相容性。方法:①实验分6组:对照组(未接触镍钛合金的血液)、未包被组、Chi组和Chi/r-Hir低浓度、中浓度和高浓度组;②血液相容性实验:包括血液溶血试验和动态全人血接触试验。观察实验各组镍钛合金片溶解红细胞的特性;检测动态全人血接触后实验各组血液APTT、PT和TT,观察其抗血栓性能;扫描电子显微镜观察动态全人血接触后实验各组镍钛合金片表面血小板黏附、聚集和纤维蛋白吸附情况。③细胞相容性实验:将体外培养人脐静脉内皮细胞株(HUVECs)种植于实验各组镍钛合金表面,孵育72h,分别应用Fn和Ki67免疫荧光抗体标记,在荧光显微镜下观察HUVECs在镍钛合金表面的黏附、增殖情况并进行半定量分析。结果:①实验各组溶血率均小于1.7%;②动态全人血接触后,实验各组红细胞、白细胞、血小板计数均在正常值范围内且组间未见差异;与未包被组和Chi组相比,Chi/r-Hir包被明显延长APTT和TT(P<0.001);Chi/r-Hir包被延长APTT和TT作用与重组水蛭素浓度有关,低、中、高浓度组间相比存在差异(方差不齐采用Dunnetts法P<0.05);③扫描电镜结果显示,Chi/r-Hir组与Chi组相比,镍钛合金表面血小板黏附、聚集和纤维蛋白吸附明显减少。④细胞相容性实验:经过72h孵育,镍钛合金片边缘细胞生长和移行良好。Fn荧光标记观察HUVECs在镍钛合金片表面黏附依次为:Chi组>未包被组>Chi/r-Hir组,Chi/r-Hir包被不同浓度组间HUVECs黏附未见差异;Ki67免疫荧光标记观察HUVECs在镍钛合金表面增殖依次为:未包被组=Chi组>Chi/r-Hir组,Chi/r-Hir包被不同浓度组间HUVECs增殖未见差异。结论:Chi/r-Hir包被先天性心脏病介入封堵材料镍钛合金后,具有良好抗凝性能,但不利于HUVECs黏附和增殖。
OBJECTIVE: To evaluate the biocompatibility of chitosan / recombinant hirudin (Chi / r-Hir) covalently coated with nitinol alloy for congenital heart disease interventional blocking material. Methods: ① The experiment was divided into 6 groups: control group (untreated with nitinol), uncoated group, Chi group and Chi / r-Hir low, medium and high concentration group; ② blood compatibility test: Including blood hemolysis test and dynamic whole human blood exposure test. The characteristics of lytic cells in each group were observed. The APTT, PT and TT were measured in all groups after dynamic whole human blood exposure. The anti-thrombotic properties were observed by scanning electron microscopy (TEM) Titanium plate surface platelet adhesion, aggregation and fibrin adsorption. ③ Cell compatibility experiment: Human umbilical vein endothelial cell line (HUVECs) were cultured in vitro on each group of Ni-Ti alloy surface and incubated for 72h. Fn and Ki67 immunofluorescence antibody labeling was used respectively. HUVECs were observed under a fluorescence microscope in nickel Titanium alloy surface adhesion, proliferation and semi-quantitative analysis. Results: ① The hemolysis rates of all groups were less than 1.7%; ② After the whole-body blood exposure, the counts of erythrocytes, white blood cells and platelets in the experimental groups were within the normal range and no difference was found among the groups; Chi / r-Hir coated group was significantly prolonged APTT and TT (P <0.001); Chi / r-Hir coated prolonged APTT and TT effect and recombinant hirudin concentration related to low, medium and high concentrations of interphase (P <0.05 by Dunnetts method); (3) The results of scanning electron microscopy showed that the adhesion and aggregation of platelets and the adsorption of fibrin in the Ni / Ti alloy were significantly reduced in the Chi / r-Hir group compared with the Chi group. ④ cell compatibility experiments: after 72h incubation, nitinol edge cells grew well and migrate well. The adhesion of HUVECs to NiTi plates was observed by Fn fluorescent labeling in the following order: Chi group> uncoated group> Chi / r-Hir group, no difference was observed between Chi / r-Hir coated HUVECs and Ki67 immunofluorescence The proliferation of HUVECs on NiTi alloy surface was observed as follows: uncoated group = Chi group> Chi / r-Hir group. No difference was observed in the proliferation of HUVECs between different concentrations of Chi / r-Hir coated group. Conclusion: Chi / r-Hir coated with nitinol coated congenital heart disease has good anticoagulant properties, but not conducive to HUVECs adhesion and proliferation.