基于MRI/NIRF成像技术评估CTGF对动脉粥样硬化易损斑块稳定性影响的研究

Connective tissue growth factor (CTGF) is one of the major cytokines involved in the extracellular matrix synthesis. But the role of CTGF in unstable vulnerable plaques remains controversial, plaque rupture is promoted by metalloproteinases (MMPs). In this project, we seek to develop the novel multifunctional nanoparticle probes targeted CTGF and MMP-2. The probe design involves linking Cy5.5-MMP substrate to PEG conjugated superparamagnetic Fe3O4 nanoparticles and conjugated with anti CTGF single chain Fv antibody fragments(ScFv). This CTGF-targeted molecular imaging probes show protease specific fluorescence recovery caused from the specific peptide cleavage by MMP-2. It has potential as an 'activatable' dual imaging probe for unstable plaque. .We will explore the relationship between the development of unstable vulnerable plaques and the expression of CTGF and enzymatically active MMP-2 in vulnerable regions of unstable carotid plaques. An animal model with disruptions of atherosclerotic plaques is induced in the left carotid artery of apoE-/- mice by placement of a constricting collar plus stress- and LPS-costimulated. The effect of a CTGF-neutralizing monoclonal antibody and a MMP-2 inhibitor for the prevention of atherosclerotic plaque rupture will be assessed in apoE-/- mice，the dual imaging probe will be used to detect CTGF expression and MMP-2 enzymatically active in vivo using both NIRF optical and MR imaging. Histological assessment of iron oxide deposition by Perl staining and immunohistochemical assessment of smooth muscle cells, macrophages, endothelial cells, MMP-2 will be performed within the atherosclerotic lesions in the left carotid artery. We try to clarify the new mechanism of vulnerable plaques formation. CTGF-promoted vulnerable plaques formation is mediated by MMP-2. The use of anti CTGFScFv-Fe3O4-MMPCy5.5 to target MMP-2 and CTGF has the potential to be developed into an effective molecular imaging agent to monitor vulnerable plaques.

结缔组织生长因子(CTGF)是调节基质合成的重要生长因子，其在致动脉粥样硬化不稳定斑块的作用不明。而基质金属蛋白酶(MMPs)可导致斑块不稳定。本项目拟以CTGF、MMPs为成像靶点，将聚乙二醇修饰的超顺磁性氧化铁纳米粒与近红外荧光(NIRF)Cy5.5MMP底物肽桥联合成可控荧光的纳米颗粒Fe3O4-MMP，并耦联抗CTGF单链抗体(ScFv)，构建抗CTGFScFv-Fe3O4-MMPCy5.5双靶向分子探针。建立ApoE基因敲除鼠颈动脉不稳定斑块模型，以CTGF中和单抗阻断及MMP靶向药抑制斑块CTGF及MMP的表达，通过该探针在小鼠不稳定斑块的MRI和NIRF双重显像，研究CTGF在不稳定斑块中的作用及其与MMP活化的关系。同时应用血管病理学及分子生物学等方法验证影像变化，探讨CTGF在易损斑块靶向分子成像的价值，建立活体特异性分子影像学评价体系。以实现对不稳定斑块的早期诊断。

由动脉粥样硬化（AS）斑块引起的心脑血管事件是全球首要死亡原因，斑块破裂与否主要取决于AS斑块的稳定性，先进的影像技术能早期判断斑块的性质而预测远期不良事件，为心脑血管事件的预测和预防提供了光明的前景。该项目以结缔组织生长因子(CTGF)、金属基质蛋白酶（MMPs）、叶酸受体（FR）等为成像靶点，构建多种针对AS斑块中巨噬细胞的分子探针，采用MRI、PET及超声光电等多模态成像活体成像，动态观察斑块形成，研究巨噬细胞多种标记物分子成像的意义。建立 ApoE 基因敲除鼠颈动脉AS斑块模型，研究 CTGF 在AS斑块中的作用及其与 MMP 活化的关系。同时应用血管病理学及分子生物学等方法验证影像变化，探讨 CTGF、OxLDL、FR等靶标在易损斑块靶向分子成像的价值，建立活体特异性分子影像学评价体系。结果显示:使用低剪切力造成的小鼠颈动脉斑块模型，CTGF抗体-氧化铁纳米颗粒（anti-CTGF-USPIOs）能识别CTGF高表达的巨噬细胞而对AS斑块成像，该探针与CTGF及巨噬细胞共定位。Anti-CTGF腹腔内注射能显著抑制AS斑块的增长，抑制MMP2的活性。CTGF在AS斑块行成中起重要作用，是AS成像和治疗的重要靶标。同时我们也构建了anti-Ox-LDL-USPIO, FR靶向USPIO等MRI巨噬细胞靶向造影剂活体对腹主动脉、颈动脉AS斑块成像。构建以黑色素纳米颗粒（MNPs）为基础，连接靶向αvβ3 的RGD环肽，建立具有靶向巨噬细胞效应多模态小分子探针平台，将MNP-RGD连接89Zr，合成PET探针89Zr-RGD-MNP，89Zr-RGD-MNP可活体靶向小鼠颈AS斑块，其作用可被阻断剂阻断。通过本课题，我们建立了活体颈动脉斑块检测多模态体系，采用MRI、PET及超声光电等成像方式对巨噬细胞活体成像。建立活体特异性分子影像学评价体系。以实现对不稳定斑块的早期诊断。

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