靶向超声造影微泡介导Bcl-2和Ang-1联合基因治疗急性冠状动脉综合症的实验研究

The acute coronary syndromes, including unstable angina and acute myocardial infarction, currently constitute a major preoccupation of clinical cardiology. Initial therapy for acute coronary syndrome should focus on restoring blood flow to sites beyond hemodynamically significant stenoses in the coronary arteries. The revascularization therapies will effectively relieve angina pectoris in many cases. Gene therapy is a promising approach for achieving myocardium revascularization in coronary heart disease. In previous study, we have constructed targeted microbubbles which can combine with E-selectin or ICAM-1 specifically. More significantly, we synthesized a novel targeted microbubble with 12-mer peptide on the surface with specific E-selectin affinity for the first time in the world. And we completed the in vivo study of targeted imaging of ischemic myocardial tissue after ischemia/reperfusion injury and proved that the targeted microbubbles can be used to detect the ischemic myocardial tissue on early stage specifically and sensitively. Based on our previous work, we hypothesize that we can achieve the therapy goal at the same time when we perform targeted ultrasound molecular imaging using microbubble loaded with the targted legand of ICAM-1 on its surface and carry the therapeutic genes (Bcl-2 and Ang-1) inside. Ultrasound-targeted microbubble destruction (UTMD) is a promising nonviral method for targeting gene therapy that may be useful in ACS targeted therapy. We will relieve Bcl-2 and Ang-1 at the myocardial infarction using UTMD technique in rat myocardial infarction and may provide reliable experimental proof for the targeted gene therapy of acute coronary syndrome.

急性冠状动脉综合症(Acute Coronary Syndrome, ACS) 早期治疗的关键是挽救缺血心肌，缩小心梗面积，甚至避免心肌梗死的发生，大量研究表明促血管生成的基因治疗已逐渐成为治疗冠心病极有应用前景的治疗方法。课题申请人在前期工作中成功合成ICAM-1、E-选择素单靶向及联合靶向微泡，并在世界范围内首次采用多肽（AF10166）作为配体成功制备E-选择素靶向微泡，证明了靶向超声分子成像技术在大鼠心肌缺血早期（15min-4h内）即可敏感地检测出心肌组织缺血。本课题拟在已完成的ACS靶向成像的基础上，利用目前已成功合成的靶向微泡与缺血心肌特异性结合这一特性，进一步在造影剂内加入靶向治疗基因，构建携有Bcl-2和Ang-1目的基因的靶向超声微泡，并用超声破坏载基因微泡转染细胞的体外实验进行验证，为ACS的靶向基因治疗提供有力的实验依据。

急性冠脉综合征（Acute Coronary Syndrome, ACS）是一组由急性心肌缺血引起的临床综合征，是导致冠心病患者死亡的主要原因，其早期诊断及早期干预治疗具有巨大的临床意义。ACS 早期治疗的关键是挽救缺血心肌，缩小心梗面积，甚至避免心肌梗死的发生，大量研究表明促血管生成基因治疗通过引入外源性生长因子促进缺血区新生血管和侧枝循环形成，从而改善心肌缺血状况，已逐渐成为治疗冠心病极有应用前景的治疗方法。超声靶向破坏微泡定位释放技术（Ultrasound-targeted microbubble destruction，UTMD）为基因载体及基因导入方式的选择开辟了一条新的渠道，其在保障转染的安全性的基础上也提高了转染效率。而靶向超声分子成像技术利用靶向超声微泡与目标分子特异性结合，产生持续性对比增强效应从而对缺血心肌的部位及范围作出定位。本课题在已完成的急性冠脉综合征靶向成像的基础上，利用目前已成功合成的ICAM-1靶向微泡与缺血心肌特异性结合这一特征，进一步在造影剂内加入靶向治疗基因，构建携有Ang-1治疗基因的靶向超声微泡，并借助超声靶向破坏微泡定位释放技术局部爆破微泡，释放治疗基因，从而进行靶向治疗。本课题成功地构建了含人Ang-1基因的pEGFP-N3质粒并分离培养了大鼠心脏微血管内皮细胞；成功地构建了纳米级携Ang-1质粒的ICAM-1靶向微泡，其平均直径为226±23nm，同时携带Ang-1重组质粒及ICAM-1抗体的携带率达到69.8%。本研究中，在确保细胞存活率的基础上提高转染效率对超声辐照参数进行了反复的验证，最终优化了超声辐照参数。我们将大鼠心脏微血管内皮细胞进行厌氧培养模拟心肌缺血缺氧过程，应用UTMD技术介导纳米级携Ang-1质粒的ICAM-1靶向微泡转染细胞证明了： UTMD技术可以促进基因的转染，UTMD联合靶向微泡可以大大提高基因载体及基因的转染效率，为ACS的靶向基因治疗提供有力的实验依据。.本课题的研究成果共有三篇SCI论文，其中一篇已发表，影响因子3.408，另外两篇已接收，影响因子分别为4.428和1.532。另有一篇中文核心期刊文章已被中国医学影像技术接收。共培养硕士研究生2名，协助培养博士研究生1名。