心血管内靶向定位基因递送体系—载基因支架的实验研究

发布时间：2018-08-02 12:04

【摘要】：经皮腔内冠状动脉成形术(Percutaneous Transluminal Coronary Angioplasty，PTCA)是冠状动脉硬化性心脏病的主要治疗手段，但PTCA术后血管再狭窄的发生率高达15—60％，迄今仍是临床亟待解决的难题。转基因技术的飞速发展为血管再狭窄的基因治疗奠定了基础。心血管内基因治疗的成功必须依靠有效的治疗基因，，安全的载体和可以把基因(和载体)递送到血管内靶部位的递送体系。心血管内基因治疗的特殊性在于很难把基因专一性递送到血管组织而不进入血液循环系统。以往的研究大多数采用球囊导管向血管内灌注基因(和载体)，研究表明，灌注到血管内的载体大部分随血流进入全身循环系统，到达病灶局部的基因很少，达不到治疗效果。血管内支架携带基因有其独特的优势，可以在植入支架的同时将基因递送到心血管内的病灶部位，借助支架与血管壁的紧密接触，使基因被局部血管组织吸收，随着基因的缓慢释放，达到长期的基因转染和表达。基因载体分为病毒载体和非病毒载体。病毒载体转染效率高，但安全性方面存在潜在的危险性。非病毒载体安全性好，易于制备，近年来倍受关注。阳离子脂质体和壳聚糖是目前研究最广泛的非病毒载体，显示出了一定的优越性。本课题针对心血管基因治疗的主要技术难题，采用血管支架为基因递送平台，将抗DNA抗体—质粒DNA—阳离子脂质体三元复合纳米基因载体和壳聚糖基因纳米粒两种非病毒载体结合在支架上，并通过体内外试验验证这一新型血管内基因递送体系的有效性和可行性。本文第一章对PTCA术后再狭窄的发病机理及治疗现状进行了综述。本文具体研究内容如下： 1．制备了新型抗DNA抗体—质粒DNA—阳离子脂质体三元复合纳米基因载体(DAC)，筛选出了最佳配方，并初步进行了细胞摄取及细胞基因转染实验。结果发现，抗DNA抗体—质粒DNA—阳离子脂质体可以自组装为360nm左右的球形粒子，与传统的质粒DNA—阳离子脂质体二元基因载体(DC组)相比，DAC体系对质粒DNA(28.5±1.3％vs13.6±0.9％，p＜0.01)的包封率明显增加，并且明显地提高了细胞基因转染效率(41.8％vs10.2％，p＜0.01)。采用双重荧光标记联合共聚焦显微观察发现抗DNA抗体可促进质粒DNA进入细胞核。使用胶原对金属支架表面进行涂层，通过化学和免疫双重偶联的方法将上述DAC载体固定化在支架表面。使用放射性同位素分别标记抗DNA抗体和质粒DNA来测定支架结合量及释放曲线。用细胞培养及动物体内植入实验验证了这一新型基因递送体系的转基因效果。结果显示，支架表面通过化学交联的胶原涂层具有很好的均一性
[Abstract]:Percutaneous transluminal coronary angioplasty (Percutaneous Transluminal Coronary) is the main treatment for coronary sclerosing heart disease, but the incidence of restenosis after PTCA is as high as 15-60, which is still a difficult problem to be solved. The rapid development of transgenic technology has laid the foundation for gene therapy of vascular restenosis. The success of gene therapy in cardiovascular system must depend on effective therapeutic genes, safe vectors and delivery systems that can deliver genes (and vectors) to target sites in blood vessels. The particularity of gene therapy in cardiovascular system is that it is difficult to transfer gene specifically to vascular tissue without entering the circulatory system. Previous studies have mostly used balloon catheters to infuse genes (and vectors) into blood vessels. It has been shown that most of the carriers infused into the blood vessels enter the systemic circulatory system with the blood flow, and there are few genes to the local focus, which can not achieve the therapeutic effect. Endovascular stents carry genes with unique advantages. They can be delivered to the focus of the cardiovascular system while stents are implanted, and the genes can be absorbed by the local vascular tissues by the close contact between the stents and the vascular walls. With the slow release of genes, long-term gene transfection and expression are achieved. Gene vectors are divided into viral vectors and non-viral vectors. The transfection efficiency of virus vector is high, but there is potential danger in safety. Non-viral vectors are safe and easy to be prepared, and have attracted much attention in recent years. Cationic liposomes and chitosan are the most widely studied non-viral carriers, showing some advantages. In view of the main technical problems of cardiovascular gene therapy, the vascular stent is used as the gene delivery platform. DNA antibody plasmid DNA-cationic liposome ternary composite nano-gene vector and chitosan gene nanoparticles were combined on the scaffold. The validity and feasibility of this new intravascular gene delivery system were verified by in vivo and in vitro experiments. The first chapter reviews the pathogenesis and treatment of restenosis after PTCA. The main contents of this paper are as follows: 1. A novel DNA antibody plasmid DNA-cationic liposome ternary nano-gene vector (DAC), was prepared. The best formula was selected, Cell uptake and gene transfection were also studied. The results showed that DNA antibody plasmid DNA-cationic liposomes could self-assemble into spherical particles about 360nm. Compared with the traditional plasmid DNA-cationic liposome binary gene vector (DC group), the encapsulation efficiency of plasmid DNA (28.5 卤1.3%vs13.6 卤0.9p < 0. 01) was significantly increased, and the efficiency of gene transfection was significantly improved (41.8 vs 10. 2% P < 0. 01). Double fluorescent labeling and confocal microscopy showed that anti DNA antibody could promote plasmid DNA into nucleus. The metal scaffold was coated with collagen and immobilized on the scaffold by chemical and immune coupling. Radioisotopes were used to label anti DNA antibody and plasmid DNA respectively to determine the binding capacity and release curve of the scaffold. The transgenic effect of this novel gene delivery system was verified by cell culture and animal implantation in vivo. The results showed that the surface of the scaffold was well homogenized by chemically crosslinked collagen coating.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

【参考文献】