【作者】 周忠江；

【导师】 刘伊丽； 吴平生；

【作者基本信息】 第一军医大学 ， 内科学心血管病， 2002， 博士

【摘要】 一．前言 1．研究背景 冠心病是世界范围内公认的威胁人类身心健康及引起死亡的第一杀手，冠心病的实质是缺血和缺氧。近二十年来，人们不断努力，取得了许多成就，循证医学指导下的新的更为合理的心血管药物以及以冠状动脉旁路搭桥术（CABG）和血管成形术（PTCA）为代表的血运重建心肌再灌注疗法，使冠心病尤其是急性心肌梗死的治疗和预后取得了长足的进步。然而，仍有诸多重要问题未予解决。例如，对于一些由于自身冠状动脉病变复杂、严重、弥散、终末小分枝病变、以及合并其它不适合介入治疗的全身性疾病如糖尿病、肿瘤等，则不能进行PTCA和CABG冠脉重建；有些病人虽可进行冠脉重建，但重建后血供仍不充分，缺血心肌濒临坏死、凋亡、纤维化；另外PTCA和CABG术后再狭窄问题，使心肌再灌注疗法面临挑战。因此，寻求其它血运重建策略迫在眉睫，人们寄厚望于称为“分子搭桥”的治疗性血管新生（Therapeutic angiogensis）以及心肌干细胞移植。 缺氧心肌微循环的代偿包括微血管扩张、征募储备血管、微血管新生及动、静脉侧支形成，严重冠心病患者即使在合并使用硝酸酯和钙拮抗剂等药物治疗基础上，内源性的促血管生长调节下的微循环代偿已达极限。因此，在药物治疗、血运重建基础上，通过输入外源性的促血管生长因子，促进心肌血管新生（Therapeutic Angiogenesis）、改善心肌缺血即心肌血管分子搭桥，是人们正在努力探索的研究热点。研究证实，Angiogenesis是由多种有丝分裂原相互作用、调控的瀑布式生物反应，一系列血管生长的促进和拮抗因子，如VEGF（血管内皮生因子）、FGF（纤维生长因子）、Angiopoietin（血管生长素）、Ephrins、Thrombospondin-1（凝血栓蛋白）等，通过血管内皮高亲合力受体及信号传导途径参与血管生成。分子生物学在心血管领域的应用及心肌分子搭桥基因治疗的飞速发展，使分子搭桥已进入临床进行初步实践；如开胸心肌注射VEGF₁₂₁重组腺病毒、经心内膜和心包注射重组VEGF₁₆₅和VEGF₁₂₁真核表达质粒、冠脉内注射bFGF、VEGF16。进行血运重建的临床试验等。这些研究虽然取得了一些令人振奋的效果，但有创、高风险的基因传输方式明显限制了分子搭桥疗法的深入开展。 超声在基因和活性药物靶向传输中的应用是正在兴起的研究热点，。其核心是应用超声波及声学造影剂微气泡发展基因和药物定向释放技术，即超声介导微泡空化靶向传输系统。该系统的原理是将声学造影剂微气泡耦连或包载靶向药物或基因，使载药微泡在显影心肌的同时，利用声学造影剂微气泡在超声场内发生的空化效应，将基因和药物运载和释放到特定的组织和器官，使局部组织达到有效的治疗浓度。己有充分研究证实该系统的可行性和应用前景，其全身给药少、局部浓度高、无创、简便、并可在床边进行等优点，赋予当前靶向性差、正处于低迷状态的基因疗法以新的希望。2．目的和意义 本研究拟以上述理论、研究背景为依据，利用分子生物学、细胞生物学及声学造影技术，以蛋白质靶向微泡在组织水平探索超声介导微泡空化靶向传输放射性核素标记的大分子物质BSA的可行性，以蛋白质微泡。脂质体微泡在细胞水平初步探索超声介导微泡空化效应促内皮细胞报告基因转染、表达的可能性；以自制脂质体靶向微泡向大鼠梗塞心肌靶向传输自行构建、克隆的血管内皮生长因子基因VEGF165，并在心肌梗塞动物模型评价VEGF16；分子搭桥血管新生的效果：为超声介导微泡空化靶向传输系统的深入研究以及冠心病分子搭桥基因治疗服务于临床，奠定理论基础和提供实践经验。 第一军医大学南方医院率先研制的第一。二代造影剂填补了国内心肌声学造影的一项空白，如果研制成功能进行心肌靶向传输的第三代造影剂，则能紧跟国际前沿，进一步丰富超声介导微泡空化靶向传输系统这一涉及多个学科的复杂理论体系。未来超声介导微泡空化靶向传输系统的成功实践，不但会给心肌血管新生的分子搭桥基因治疗提供有效的基因靶向投送手段，而且还有助于攻克目前基因治疗靶向性差的世界难题。同时，靶向微泡的成功研制还将用于诸如肿瘤、外用血栓病的治疗，因此本研究具有重要的经济和社会意义。3．研究内容 一6－ 本课题主要进行以下几个方面的的研究： ＊)在国内应用超声介导微泡空化靶向传输的动物及细胞模型，首次进行 靶向传输研究； 。c)向大鼠心肌传输放射性核素标记的牛血清白蛋白，取得预期心肌靶向 一传输效果； m 向培养的人血管内皮细胞传输LacZ报告基因，证明脂质体靶向微泡 有良好的靶向传输及促基因转染、表达效果； O) 向大鼠梗塞心肌传输自行构建的血管内皮生长因子基因VEGF165；取 得一定的促血管生成效应： 门在血管内皮生长因于基因表达载体的构建过程中，发现一新的血管内 皮生长因子基因的剪接形式更多还原

【Abstract】 Introduction 1. BackgroundCoronary Heart Disease (CHD),which essence is ischmia and hxpoxia,is publicly recognized as main threat for human health and cause the leading death worldwide. With the guidance of evidence medicine,much achievement have been made on the treatment of CAD,especially acute myocardial infarction over the past decades due to lots of newly-found drugs and the reperfusion therapies represented by coronary artery bypass graft (CABG) and percutaneous transluminal coronary angioplasty (PTCA);however,a lot of issues remain resolved. A growing number of patients neither are suitable for candidates for conventional intervention therapies secondary to anatomical constraints imposed by the severity or extent of their coronary artery disease and some systematical condition such as diabetes and tumor,nor can they get enough blood supply under intervention. If such condition developed,the ischemic myocardium would be doomed to apoptosis,necrosis and fibrosis. It’s urgent for people to seek other biological revascularization strategy,and a large body of work involved in both therapeutic angiogensis and stem cell graft are hopefully under way.Normally microcirculation compensation of ischemic heart includes microvessel enlargement,angiogensis and collateral vessel development between artery and vein. However,even nitrates and calcium antagonist were fully administrated,intrinsic microcirculation adjustment still could not reach beyond the basic demand of oxygen and blood of ischemic myocardium,therefore therapeutic angiogenesis induced by exogenesis growth factor which can initiate the formation of a plexus of collateral vessels in zones of ischemic myocardium serve to relieve angina and to improve advanced myocardial ischemia at the base of drug therapy and revascularization intervention. Angiogenesis,which is called "molecular graft of vessels",is a complex and cascade process that involves in stimulation of endothelial cell proliferation and migration by a huge amount of antagonist and agonistgrowth factors,including the fibroblast growth factor (FGF) family,vascular endothelial growth factor (VEGF) family,angiopoietin,ephrins and thrombospondin-1 through stimulating high affinity receptors and signal transduction passway of endothelial cells,and those mitogen factors and cascades seems to act in a coordinated way to achieve this process. The vascular endothelial growth factor(VEGF) was identified as endothelial cell specific mitogen. It has been shown to be involved in endothelial cell proliferation and blood vessel formation. VEGF mediates its function through its specific receptor on the vascular endothelial cell surface. VEGF plays important roles in variety of physiological and pathological processes including embryogenesis,tumor and some cardiovascular diseases. The rapid development of molecular biology in myocardiovascular scope and growth factor therapy offers to clinical practices. Although several clinical trials involved various endovascular delivery devices have been designed for angiogenesis,such as bolus injection of naked plasmid DNA encoding phVEGF165,phVEGF121 and bFGF of recombinated virus,either in pericardium,direct myocardium at the time of thoracotomy or intracoronary injection and endoluminal delivering,the limitations of such delivery including the possibility of increased vascular trauma,high risk,low efficiency of localization,inconsistency of delivery,and rapid washout of the agents from the vascular wall after delivery prevent the progres of further development of angiogenesis.Ultrasound,which has been playing a very important role in targeted delivering of gene and active agents,merits further exploration. The aim is to use ultrasound and ultrasound echo contrast agents to offer a targeting delivering procedure,called the delivery system of ultrasound-mediated destruction and cavitation of targeted microbubbles. The core of the approach,which depend on the cavitation effect of microbubble under ultrasound field,is locally delivered pharmacological drugs or t更多还原