基于POSS的逐级靶向基因递送系统促进血管内皮细胞增殖研究

The rapid endothelialization on small diameter artificial blood vessel lumen plays an important role, which can prevent thrombosis formation and intimal hyperplasia, and improve the long-term patency rate of artificial blood vessels. However, owing to lack of selective proliferation of endothelial cells (ECs), it is urgent to develop the POSS based, sequentially targeting gene delivery systems to promote vascular ECs proliferation. How gene complexes can be specifically selective recognized and uptaken by vascular ECs, how to enhance endosomal/lysosomal escape, and how to be localized in the nucleus, are the key scientific problems for gene transfection of vascular ECs. In this proposal, we intend to develop the multifunctionally and sequentially targeting gene delivery systems based on POSS with the aim to enhance transfection efficiency and proliferation of vascular ECs. Firstly, targeting peptides and cell-penetrating peptides will be introduced onto POSS gene carriers. These peptides can provide gene delivery systems with selective recognition ability of vascular ECs and high cell uptake ability. Secondly, charge conversional shielding system facilitates and promotes endosomal/lysosomal escape. Thirdly, genes can be effectively targeted and delivered into the nucleus of vascular ECs by nuclear localization signal peptides. These multifunctionally and sequentially targeting gene delivery systems can significantly promote the transfection efficiency and proliferation of vascular ECs by their multifunctional and sequential functions. By using the transferred ECs by sequentially targeting gene delivery systems to modify artificial vascular grafts, the rapid endothelialization of the surface of artificial vascular grafts will be enhanced, as well as the long-term patency rate can be improved. This proposal provides a new idea and approach for gene transfection of ECs, which is beneficial to solve the low endothelialization problem on the artificial vascular grafts. Thus it has important scientific significance and application value.

促进小口径人工血管内腔快速内皮化可以防止血栓形成和内膜增生，提高人工血管长期通畅率，然而，受限于血管内皮细胞选择性增殖效果不理想，亟需研究基于POSS的逐级靶向基因递送系统促进血管内皮细胞转染和增殖。如何逐级实现对血管内皮细胞特异性识别而被摄取、促进内涵体逃逸和核定位是基因转染中所面临的关键科学问题。本项目拟以POSS为核构建星型多功能基因递送系统，首先在POSS上引入靶向性和细胞穿透肽，赋予基因载体对血管内皮细胞的特异性识别能力和提高细胞摄取；其次，电荷反转层促进内涵体/溶酶体逃逸；最终，携带基因的核定位信号肽将高效地递送目的基因进入细胞核，从而显著地提升内皮细胞的转染和增殖能力。利用逐级靶向基因递送系统促进人工血管表面的快速内皮化，提高长期通畅率。本项目为血管内皮细胞转染研究提供一种新思路和技术途径，有望从根本上解决人工血管内皮化水平低的问题，具有重要的科学意义和研究价值。

促进小口径人工血管内腔快速内皮化能够防止血栓形成和内膜增生，提高人工血管长期通畅率，然而，受限于血管内皮细胞选择性增殖效果不理想，亟需研究新型基因递送系统，实现促进血管内皮细胞高效转染和增殖的目的。为此，我们研究了多功能、逐级靶向性基因递送系统，逐级实现对血管内皮细胞特异性识别而被选择性摄取、促进内涵体逃逸和核定位，从而促进血管内皮细胞转染和增殖。这种基因载体克服了内皮细胞转染中所面临的一些关键科学问题。本项目首先研究了REDV肽、CAG肽、细胞穿透肽、核定位信号肽等对基因载体的选择性递送基因的作用，设计和构建了逐级靶向基因递送多肽序列。以笼型聚倍半硅氧烷（POSS）为核，将多肽键接而构建出了星型多功能基因递送系统。在POSS上引入靶向性和细胞穿透肽，赋予基因载体对血管内皮细胞的特异性识别能力和提高细胞摄取；电荷反转层促进内涵体/溶酶体逃逸；核定位信号肽将高效地递送目的基因进入细胞核，从而显著地提升内皮细胞的选择性转染和增殖能力，促进血管形成。利用逐级靶向基因递送系统实现了人工血管表面的快速内皮化，这将有利于提高其长期通畅率。本项目有望从根本上解决人工血管内皮化水平低的问题，为血液接触植入物的研制提供新的基因递送技术，在生物材料与组织工程研究领域具有应用前景，该研究具有重要的科学意义和应用价值。发表论文合计39篇，参加国际会议和国内学术会议摘要或论文10 篇，申请发明专利16件，授权发明专利1件。培养研究生合计10名（博士5名，硕士5 名），其中培养了1 名外国留学生博士和3 名外国留学生硕士。