损伤血管靶向的多功能聚阳离子/双基因系统的构建及对术后再狭窄的防治与机制研究

Gene therapy for restenosis (RS) is still hindered by the complex pathogenesis of RS and the low efficiency of delivery systems. How to optimize the therapeutic gene and its carrier performance is a key problem for improving the treatment outcomes of RS. Our previous studies indicated that polyamidoamine-polyethylenimine (PAMAM-PEI, PME) copolymers showed high transfection efficiency and safety in the presence of serum. According to the pathogenesis of RS and the performance of PME, in this study we plan to prepare multifunctional PME/dual-gene therapy systems for preventing RS. In these systems, dexamethasone (DE) modified polyrotaxane or poly(L-glutamine) will be used as backbones for PME, PEG-antibody conjugate as a target group for recognizing injured blood vessels, nuclear signal peptide and DE as target groups for nuclear and VEGF165-TIMP3 as multi-target therapy genes. These systems have high in vivo stability, biocompatibility, anti-inflammatory effect and can efficiently and specifically deliver therapeutic gene to injured blood vessels and primary vascular cells, leading to high expression. These systems can prevent RS through accelerating endothelialization，inhibiting the activity of metalloproteinaseses, inhibiting vascular smooth muscle cells proliferation and migration and promoting their apoptosis. Present study will first optimize the performance of these therapy systems and then evaluate their effects and mechanism of preventing RS on animal model. The results of this project will provide a potential method and evidences for gene therapy of RS after percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) surgery.

发病机理复杂和传递系统低效是基因防治血管再狭窄（RS）的障碍，如何优化治疗基因及其载体性能是有效防治RS亟需解决的难题。前期研究发现核壳结构的PAMAM-PEI（PME）共聚物在血清中具有高转染活力与安全性。为此，本课题结合RS机理及PME性能，利用地塞米松（DE）修饰的聚轮烷或聚谷氨酸肽作为PME的骨架、PEG-抗体作为靶向基团、核信号肽和DE作为核导向、VEGF165-TIMP3作多靶点治疗基因，构建一类可针对损伤血管病理特点的功能化PME/双基因RS防治系统。该系统在体内可具有稳定性高、生物兼容性强、抗炎且可将基因高效靶向传递至损伤血管及在原代细胞中高表达的功能，能够通过加速血管内皮化、抑制基质金属蛋白酶活力、阻碍血管平滑肌细胞增殖与迁移和促进其凋亡的方式对RS实现多靶点防治。研究将对系统的性能进行优化，对其体内防治效果及机制进行评估，从而可望为临床基因防治RS提供新疗法与科学依据。

发病机理复杂和传递系统低效是基因防治血管再狭窄（RS）的障碍，如何优化治疗基因及其载体性能是有效防治RS亟需解决的难题。基于聚阳离子聚合物的低毒高效的基因传递性能，本项目合成了PAMAM G0-PLG-g-PEI、PEG-PLG-PEI-De、MPEG-CD-PEI-De等聚合物，并对其结构、接枝率、功能基的种类、聚阳离子/基因的量比等方面进行了筛选和优化，以达到提高转染效率和降低毒性的目的。评价了它们的基因传递系统的物化和生物性能，并考察了载体对血管内皮细胞等原代细胞的基因传递效率。PEG-PLG-PEI-De介导VEGF-TIMP3对血管平滑肌细胞和内皮细胞均表现出较高的基因转染效率，且在有血清转染条下的效率更高，可以促进内皮细胞增殖、阻碍血管平滑肌细胞增殖与迁移并使其凋亡，可对RS实现多靶点防治。动物实验进一步表明，该基因体系可以减轻损伤血管再狭窄的程度。研究结果将有望为临床PCI或CABG术后防治RS提供新的治疗思路和途径，并对促进聚阳离子基因载体发展具有科学意义。