随着易损斑块在急性心血管事件中作用不断深入研究，早期识别易损斑块是临床面临的难题。分子影像学发展为解决该难题带来希望。但是目前尚未研发出高效、特异分子探针早期识别易损斑块。本课题前期研究发现核素标记精氨酸-精氨酸-亮氨酸（RRL）环肽序列具有特异性结合新生血管内皮细胞特点。本课题应用间接法HYNIC偶联对RRL进行放射性核素锝标记，获得新型分子探针99mTc-HYNIC-RRL，靶向易损斑块新生血管形成生物学过程，在球囊拉伤易损斑块家兔模型上，通过体内SPECT/CT功能成像，体外免疫组化及细胞结合实验，评价该探针对易损斑块活体、实时成像有效性。在细胞、分子水平通过体外细胞饱和/抑制、酶活性调节实验，研究新生血管内皮细胞高表达的基质金属蛋白酶（MMP）及信号通路与99mTc-HYNIC-RRL相互作用，探索分子探针靶向示踪易损斑块机制，为有效诊断易损斑块、预防急性心血管事件提供方法学依据。

As the vulnerable plaque play an important role in the acute cardiovascular events, how to identify vulnerable plaque early and effectively is a big clinical facing problem. Molecular functional imaging brought hopes to solve this problem. However, there are no efficient and specific molecular probe developed at present to image the vulnerable plaque. In our previous work we found that the peptide(RRL) which contained arginine-arginine-leucine key sequence could specifically combined with vascular endothelial cells. Therefore, this study radiolabeled RRL with 99mTc by HYNIC and will exam targeted effects of 99mTc-HYNIC-RRL to image neovascularization of vulnerable plaques. We will inject 99mTc-HYNIC-RRL to trace the vulnerable plaques using SPECT/CT on balloon injuried rat carotid artery injury model. And then we will test the effectiveness and specificity of 99mTc-HYNIC-RRL to combine with the vulnerable plaques by immunohistochemistry and cell experiments. Furthermore, we will research the relationship between matrix metalloproteinases and 99mTc-HYNIC-RRL by measureing the enzyme activity after intervened by RRL and then carry out saturated/inhibition experiments and finally explore the mechanism of 99mTc-HYNIC-RRL targeted to combine of vulnerable plaques. The noval peptide probe 99mTc-HYNIC-RRL could target neovascularization to image vulnerable plaques early and prevention of acute cardiovascular events.

动脉粥样硬化斑块破裂引起血栓形成是临床急性冠脉综合征的根本原因。研究发现斑块的不稳定性与斑块局部的炎症反应、新生血管密切相关。如何对易损斑块进行早期识别是避免临床心血管事件的关键。评估斑块内新生微血管及炎症反应的存在及程度将可能是预测易损斑块的重要指标。早期识别易损斑块是临床的难题。分子功能影像学的发展为解决该难题带来了希望。但是目前尚未研发出高效、特异分子探针早期识别易损斑块。本课题前期研究发现核素标记精氨酸-精氨酸-亮氨酸（RRL）环肽序列具有特异性结合新生血管内皮细胞特点。本课题应用间接法HYNIC偶联对RRL进行放射性核素锝标记，获得新型分子探针99mTc-HYNIC-RRL，经过多次核素标记实验，优化标记条件后，获得99mTc-HYNIC-RRL的最高标记率为88.4%，最佳标记条件是SnCl2（10ul，1ug）+醋酸铵（80ul）+酒石酸钠(10ul) +多肽（50ul，50ug）+Tc液（100ul，500uci）震荡摇匀后室温静置1h，总体积250ul。99mTc-HYNIC-RRL靶向结合于易损斑块新生血管内皮细胞，在球囊拉伤易损斑块家兔模型上，通过体内SPECT/CT功能成像，腹主动脉斑块部位可见放射性摄取，评价该探针对易损斑块活体、实时成像有效性，并以18F-FDG PET/CT显像作为对照。完成显像后取离体血管组织，病理学染色及免疫组化分析，CD31表达阳性，提示新生血管生成。CXCR4及CD68表达阳性，提示炎性反应过程。通过进行绿色荧光染色，Confocal激光共聚焦实验观察RRL与血管内皮细胞、血管肉瘤细胞及平滑肌等细胞的结合能力。在细胞、分子水平通过PCR及蛋白免疫印迹实验，发现新生血管内皮细胞高表达VEGFR-2，提示VEGFR-2在99mTc-HYNIC-RRL与易损斑块相互结合的过程中发挥重要的作用，MMP-9在易损斑块组织中的表达量有限，而近期研究发现的CXCR4在易损斑块组织有较多的表达，可能是99mTc-HYNIC-RRL的候选靶点。通过本项目的研究，分子探针99mTc-HYNIC-RRL应用于动脉易损斑块进行分子成像是可行的，但是有效性还有待于进一步提高，CXCR4及VEGFR-2是99mTc-HYNIC-RRL靶向结合于易损斑块的可能靶点，本研究为有效诊断易损斑块、预防急性心血管事件提供方法学依据。