【作者】 张翮；

【导师】 计剑； 任科峰；

【作者基本信息】 浙江大学 ， 高分子材料， 2018， 博士

【摘要】 基因传递是基因治疗发挥效果中至关重要的一环。基于生物材料表面介导的基因传递方式凭借其原位定域性、长效性、稳定性等优势,在诸多体内疾病治疗应用中扮演着重要角色。如何为现有表面介导基因传递手段存在的不足提出解决思路,并在生物体内具体应用中展现表面介导基因传递的优势,是该领域的重要挑战。为赋予表面基因传递方式对于癌细胞与正常细胞之间的差异化选择性转染能力、为进一步提升对于细胞的转染效率、以及为通过表面介导基因传递手段实现功能化基因在体内组织的有效传递,本文开展了如下研究:1.为通过表面介导基因传递方式实现癌细胞与正常细胞之间的差异化转染,本研究将包载基因分子的基质金属蛋白酶(MMP)敏感水凝胶通过毛细作用负载于水滴模板多孔膜表面,构建了多孔膜-水凝胶基因传递表面。该多孔膜-水凝胶基因传递表面通过孔间上表面提供细胞粘附点,通过孔内水凝胶实现MMP酶敏感刺激响应DNA释放,对于含癌细胞的细胞群体具有显著转染效果,而对正常细胞无明显转染。该表面实现了癌细胞诱导的选择性刺激响应基因传递,为构建具有刺激响应基因传递行为的医用植入介入器件表面提供了思路。2.为进一步提高表面介导基因传递体系对于细胞的转染效率,本研究采用具有近红外光热效应的聚多巴胺-聚乙烯亚胺(PDA-PEI)表面,构建了光热辅助的表面介导基因传递平台。研究表明这种光热辅助的表面介导基因传递体系不仅可通过光热效应增强DNA释放,而且可有效诱导细胞膜扰动增强细胞膜通透性。该光热辅助的表面介导基因传递平台对于难以转染的原代内皮细胞的转染效率,以及对于大尺寸质粒对细胞的转染效率均具有显著提升作用,为改善传统方法难以实现的转染过程提供了新方法。3.针对转化生长因子-β1(TGF-β1)分泌在诱导血管再狭窄过程中的重要作用,本研究通过层层组装技术将编码TGF-β1-短发夹RNA(TGF-β1-shRNA)的质粒DNA(pDNA)负载于血管支架表面,构建了特异性抑制TGF-β1分泌、抑制再狭窄的表面介导功能基因涂层。该表面能够显著转染体外培养的细胞和体内血管组织并实现对于TGF-β1分泌的调控,进一步起到了抑制细胞外基质过度分泌和新生内膜过度增生的效果。该研究显示了表面介导的基因传递手段在针对抑制支架内再狭窄这一体内具体应用中的独特优势,为心血管疾病领域抗支架内再狭窄材料的设计提供了潜在可能性。更多还原

【Abstract】 Gene delivery is the most significant part of gene therapy.Among various gene delivery strategies,surface-mediated gene delivery method plays a more and more important role in applications for disease treatment due to its local,site-specific delivery behavior and its long-term efficiency.It remains to be great challenges in this field to provide novel thoughts for improving existing surface-mediated gene delivery methods and to further demonstrate the unique advantages of surface-mediated gene delivery in specific in-vivo applications.Aiming at differentiated selective gene delivery between cancer cells and normals,improving transfection efficiency,and designing surface-mediated systems for in-vivo functional gene delivery,researches were performed with details shown in the following parts:1.Aiming at the selective gene delivery between cancer cells and normals via surface-mediated delivery,in this study,a matrix metalloproteinase(MMP)-sensitive breath figure-hydrogel(BF-MDG)surface was constructed by loading gene-containing hydrogels into the breath figure holes.The upper walls of BF-MDG were hydrogel-free areas which provided sites for cell adhesion,while the holes were filled with MMP-sensitive hydrogels which released DNA in a MMP-responsive manner and selectively transfected cells only when there were MMP-overexpressing cancer cells in the system.This study provids a method for achieving selective stimuli-responsive gene delivery,and gives new thought to the combination of stimuli-responsive gene delivery and biomedical implants.2.Aiming at further enhancing transfection efficiency,in this study,a photothermy-assisted surface-mediated gene delivery system was constructed based on polydopamine-polyethyleneimine codepositing surface(PDA-PEI).By introducing photothermal effects into the transfection process,the transfection efficiency into hard-to-transfect primary endothelial cells and the transfection efficiency of a large-size plasmid were significantly enhanced.Moreover,the up-regulation of transfection efficiency were due to facilitated DNA release and cell membrane disturbance caused by local hyperthermia.This study provides new strategies for improving transfection efficiency,especially for that of hard-to-achieve transfections via traditional methods.3.Aiming at the important role of transforming growth factor-β1(TGF-β1)in in-stent restenosis processes,this study constructed a functional gene delivery stent coating for the inhibition of in-stent restenosis,by loading plasmid DNA encoding TGF-β1-short hairpin RNA(TGF-β1-shRNA)onto the stents via layer-by-layer assembly.The polyelectrolyte films were able to transfect cells in-vitro and vessel tissue in-vivo,significantly down-regulated TGF-β1 expression,and demonstrated the effect to inhibit extracellular matrix overdeposition and neointimal hyperplasia.This study further illustrates the irreplaceable advantages of surface-mediated gene delivery for biomedical applications,and provides potential for designing cardiovascular implant materials against in-stent restenosis.更多还原