白蛋白纳米t-PA基因涂层支架的研制及其抗血栓作用

Preparation of nanoparticle gene-coated stent and its antithrombotic effect

目的制备白蛋白纳米组织型纤溶酶原激活物(t-PA)基因涂层支架,观察其预防实验兔支架内血栓形成和内膜增生的效果。方法用不锈钢支架为龙骨模拟血管基底膜主要成分(透明质酸+Ⅳ型胶原蛋白+层粘连蛋白+纤维连接蛋白),配制涂层液涂于金属支架表面;制备t-PA基因质粒白蛋白纳米粒,并将其连接于涂层表面,制备成白蛋白纳米t-PA基因涂层支架。用球囊导管损伤兔髂动脉内膜制备血管内膜增生模型,将上述涂层支架植入局部血管,并用治疗性超声辅助转染。分别于术前,以及术后1、2和4周取血检测t-PA、D-二聚体含量及凝血酶原时间的变化。体视镜和常规病理观察支架植入血管堵塞及血栓形成情况。形态测量法测量内膜厚度及面积,判断内膜增生。用免疫组织化学法检测血管壁t-PA和增殖细胞核抗原的表达。结果成功制备纳米t-PA基因涂层支架。涂层支架植入血管后可见血管壁高效表达t-PA,减少支架内血栓形成和内膜增生,伴随t-PA及D-二聚体含量的增加,凝血酶原时间无明显变化。结论 t-PA基因涂层支架可减少兔支架内血栓形成和内膜增生,而未影响全身凝血。将支架术与基因治疗同步完成,为该纳米基因涂层支架的研制及支架内血栓形成的防治提供实验基础。

Objective To prepare albumin nanoparticle tissue-type plasminogen activator （t-PA） gene-coated stent and observe its effect on prevention of in-stent thrombosis and intimal hyperplasia in rabbits. Methods Stainless steel stent was coated with the coating liquid which was prepared from the main components of vascular basement membrane （hyaluronic acid ＋ type Ⅳ collagen ＋ laminin ＋ fibronectin）. To prepare albumin nanoparticle t-PA gene-coated stent, the t-PA gene plasmid was packaged by albumin nanoparticles and then connected to the coating surface. A rabbit iliac artery intimal hyperplasia model was constructed with balloon catheter. The nanoparticle t-PA gene-coated stent was implanted into the iliac artery and t-PA gene was transfected with the aid of therapeutic ultrasound. The blood levels of prothrombin time, t-PA and D-dimer were measured before and 1, 2 and 4 weeks after operation. The vascular obstruction and in-stent thrombosis after stent implantation were observed by stereoscope and routine pathological examination. Morphometry was used to measure intimal thickness and area to determine intimal hyperplasia. The expressions of t-PA and proliferating cell nuclear antigen （PCNA） were detected by immunohistochemistry. Results The t-PA gene-coated stent was successfully prepared. The expression of t-PA protein in the implanted arteries was observed, accompanied by an increase in blood t-PA and D-dimer. There was no significant change in prothrombin time after implantation of the gene-coated stent. In-stent thrombosis and intimal hyperplasia were successfully suppressed. Conclusions The t-PA gene-coated stent can successfully inhibit in-stent thrombosis and intimal hyperplasia without affecting systemic coagulation of the rabbit. The complete synchronization of stenting and gene therapy provides the experimental basis for the development of nano-gene-coated stent and prevention of in-stent thrombosis.