双功能超声微泡联合G-CSF骨髓动员靶向缺血定位诱导侧枝形成

Well-developed coronary collaterals reduce the size of myocardial infarction, preserve left ventricular function and reduce the risk of death. So identify ischemia myocardial and to promote collateral formation may preserve left ventricular function after acute myocardial infarction .Therefore,the goal of this study is to develop a efficient collateral formation before forever myocardial death .In this study,we inject G-CSF to mobilization bone marrow stem cell of models of myocardial ischemia-reperfusion pigs, use microbubbles combined with hSDF-1α plasmid and P-selection antibody to identify early ischemia myocardial and deliver SDF-1α to induce stem cells homing to ischemia tissue to enhance angiogenesis.The study include: 1 Microbubble and DNA preparation: A bicistronic plasmid vector construct for the cotransfection of both enhanced green fluorescent protein and hSDF-1α,attachment of hSDF-1α/GFP plasmid DNA and P-selection antibody to the Microbubble; 2 Animal preparation: 48 pigs divid into 4 groups , The 4 groups of pig as follows, Group1: Echocardiographic destruction without microbubbles ;Group2: Echocardiographic destruction of microbubbles ;Group3: Echocardiographic destruction of microbubbles containing P-selection antibody;Group4: Echocardiographic destruction of microbubbles containing SDF-1α/GFP plasmid DNA and P-selection antibody ; 3 myocardial ischemia-reperfusion model :48 pigs inject G-CSF for 5 days ,at 5 days later,the anterior descending coronary artery was occluded for 15 minutes, twenty minutes after reperfusion, microbubbles were intravenously inject and the triggering interval allow complete replenishment of the microbubbles under real time three- dimentional echocardialgraphy ; 4 acute myocardal infarction model,4 weeks later coronary angiography will perform to identifiy collateral formation , The anterior descending coronary will reocclud to make acute myocardial infarction model ,1week and 1month later to eveluate LVEF and LVDd, The pids will kill at 1 month after acute myocardial infarction , the heart will remove to detect expression of the transgene and infarcted area.The aim of this study is to explore the feasibility of therapeutic induce collateral formation by ultrasound-targeted destruction of microbubbles at early myocardial ischemia and to evaluate its therapeutic efficacy.

侧枝循环可以缩小心肌梗死后坏死心肌面积，在出现无法逆转心肌损伤前建立侧枝循环可能会从根本上改善急性心肌梗死预后。本研究运用携有P选择素抗体与SDF-1α质粒的双功能超声微泡心肌造影并联合G-CSF动员干细胞，超声影像识别缺血心肌并靶向趋化干细胞血管新生。①制备与SDF-1α质粒及P选择素抗体高效结合的超声微泡; ②体内造影试验优化微泡结构与功能; ③a制作小型猪急性缺血再灌注模型, G-CSF动员骨髓干细胞,静脉注射双功能微泡造影剂，超声显像识别微泡聚集缺血部位并局部声震破坏微泡释放SDF-1α质粒；b 4周后冠脉造影评价侧枝循环形成;c4周后原缺血部位制作永久性心肌梗死模型,观察心肌梗死后心功能、坏死心肌面积。本研究的创新之处在于运用超声微泡作为载体将缺血诊断与G-CSF干细胞动员促血管新生治疗相结合，在心肌发生不可逆性坏死前诱导侧枝循环形成，降低急性心肌梗死的致残、致死率。

尽管大量研究致力于内源性干细胞动员或骨髓干细胞移植促进心肌缺血后血管新生，然而众多研究结果并不理想。临床造影发现尽管冠脉病变一致，但侧枝形成能力及预后不尽相同，提示冠状动脉内皮损伤而又缺乏足够的循环干细胞归巢可能影响自身干细胞血管再生能力。故了解并促进循环干细胞功能与侧枝循环形成的关系对缺血性心脏病干细胞动员治疗的选择有着深远的意义。外周血干细胞以SDF-1/CXCR4依赖的方式到达心脏参与血管再生，CXCR4受体表达在外周血循环干细胞的迁移中扮演关键的角色，骨髓干细胞CXCR4受体的表达及心肌SDF-1表达水平直接决定了骨髓干细胞移植促血管新生的疗效。目前关于SDF-1/CXCR4轴表达与内源性侧枝形成能力的关系尚无报道。本研究发现冠脉闭塞后短期侧枝循环形成与CXCR4表达关系发现冠脉CTO病变短期内侧枝形成与外周血CD34+细胞CXCR4表达率正相关, 超声微泡作为载体可以将缺血定位与血管再生治疗结为一体，试验通过构建双分子超声微泡定位于早期缺血心肌，并释放SDF-1α趋化因子, 启动超声微泡的多种功能促进骨髓干细胞归巢血管新生以达到冠心病早期诊断及治疗的目的。通过观察已形成侧枝对心肌梗死后心功能及坏死组织面积的影响评价SDF-1/CXCR4轴在心脏血管再生意义。研究发现，外周血干细胞以SDF-1/CXCR4依赖的方式到达心脏参与血管再生，冠心病危险因素通过影响了外周血干细胞的功能进而影响了其内源性修复的效率，同时通过动物实验发现侧枝循环的存在可以缩小心肌梗死后坏死心肌面积，改善心肌缺血患者心功能，本研究给临床进一步开展干细胞治疗研究一些提示：1超声微泡作为载体可以缺血定位与血管再生治疗结为一体。2寻找宿主干细胞功能缺失的标记性因子，针对宿主干细胞功能缺失的患者有针对性地提高SDF-1/CXCR4通道效率改善心肌缺血后血管损伤修复。