基质细胞衍生因子1靶向超声对比剂在体内可与血管内皮细胞紧密结合 |
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作者姓名: | 王 梦 史 琪 穆玉明 吐尔逊那依?阿迪 |
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作者单位: | 新疆医科大学第一附属医院心脏超声诊断科,新疆维吾尔自治区乌鲁木齐市 830054 |
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基金项目: | 国家自然科学基金(81271590) |
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摘 要: | 背景:基质细胞衍生因子1是心肌梗死区域微环境中效力最强的趋化因子,在趋化干细胞修复梗死心肌以及在促进血管新生方面起到重要的作用。微泡和声学活性物质携带靶向配基,可制备成超声成像靶向对比剂并与活体细胞结合,用于分子成像,超声分子成像的关键是寻找“成像靶点”,并成功制备能与“成像靶点” 特异、高效结合的靶向超声对比剂。
目的:实验制备和评价携基质细胞衍生因子1单克隆抗体的靶向微泡超声对比剂。
方法:采用“生物素-亲和素”桥接法构建携基质细胞衍生因子1单克隆抗体的靶向微泡超声对比剂,并从外观、pH值、粒径测定、光镜及荧光显微镜下观、流式细胞仪检测等多个方面对靶向对比剂进行评价。4头中华小型猪均结扎左冠状动脉前降支第一对角支制备心肌梗死模型,2头开胸但不结扎左冠状动脉前降支第一对角支,均注入靶向超声对比剂,心肌组织冰冻切片后采用免疫荧光法检测靶向微泡的体内稳定性。
结果与结论:通过生物素-亲和素桥接法可将基质细胞衍生因子1抗体和超声微泡两者结合。体外实验中对比剂外观:表现为半透明的淡黄或绿色,静置后分层。非靶向对比剂pH值为7.02±0.12,靶向微泡对比剂的pH值为6.10±0.19。荧光显微镜下观察靶向微泡明亮且呈指环状绿色荧光环绕外壳周边,剧烈震荡后表面荧光无明显改变。靶向对比剂在携带基质细胞衍生因子1抗体之后微泡粒径大小为(2 422.62±238.82) nm。流式细胞仪检测显示,靶向对比剂在不同时间段的基质细胞衍生因子1携带率稳定,静置1 h后携带率稳定且剧烈震荡前后差异无显著性意义。在体内实验中可见靶向微泡在心梗部位血管内皮细胞处聚集。结果证实,经生物素-亲和素桥接法制备的携基质细胞衍生因子1单克隆抗体靶向微泡超声对比剂体内可与血管内皮细胞结合,在体外结合率高而且结合稳定。
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关 键 词: | 组织构建 血管内皮细胞 基质细胞衍生因子1 靶向 超声对比剂 微泡 稳定性 急性心肌梗死 分子影像学 生物素-亲和素桥接法 荧光标记 国家自然科学基金 |
Stromal cell-derived factor-1-targeted ultrasound contrast agent tightly binds to vascular endothelial cells |
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Authors: | Wang Meng Shi Qi Mu Yu-ming Tuerxunnayi Adi |
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Institution: | Department of Ultrasound, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China |
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Abstract: | BACKGROUND:Stromal cell-derived factor-1 (SDF-1) is one of the most powerful chemokines in myocardial infarction region and plays a particularly pivotal role in the homing of stem cells to an injured myocardium and promoting angiogenesis. On the other hand the microbubble and acoustics active substances carrying targetable ligands can be prepared into targeted ultrasound contrast agents that can be combined with living cells used for molecular imaging. The key of ultrasonic molecular imaging is to find “imaging targets”, and to successfully prepare targeted ultrasound contrast agent which can be combined with the imaging target specifically and efficiently.
OBJECTIVE:To prepare and evaluate targeted microbubble contrast agents with SDF-1 monoclonal antibody.
METHODS:Targeted microbubble contrast agent with SDF-1 monoclonal antibody was prepared using the
biotin-streptavidin method. The physiochemical properties of targeted microbubble contrast agent were evaluated by appearance, pH, particle diameter, optical and fluorescence microscope and flow cytometry test. Four minipigs underwent ligation of the left anterior descending coronary artery to complete the establishment of acute myocardial infarction model, and another two minipigs were subject to thoracotomy but no ligation of the coronary artery. Then, all animals were injected with microbubble contrast agents. The stability of microbubbles was assessed by immunofluorescence test in vivo.
RESULTS AND CONCLUSION:SDF-1 and microbubbles were combined by biotin-streptavidin method. In vitro appearance of the contrast agent was translucent yellow or green, and stratified after standing. pH vaule was 7.02±0.12 for non-targeted contrast agent and 6.10±0.19 for targeted microbubble contrast agent. Under the fluorescence microscope, the distribution and size of targeted microbubbles were uniform, and the microbubbles were surrounded by bright and ring shaped green fluorescence that had no changes after highly shaking. The diameter of microbubbles was (2 422.62±238.82) nm after carrying the SDF-1 antibody. Flow cytometry results showed that the carrying rate of targeted contrast agents was stable in different periods. In vivo test showed that targeted microbubbles gathered in vascular endothelial cell surface after acute myocardial infarction. These findings indicate that the targeted microbubble contrast agent carrying SDF-1 monoclonal antibody prepared by biotin-streptavidin method can be combined with vascular endothelial cells, and the binding rate is high and stable in vitro. |
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Keywords: | Tissue Engineering Ultrasonography Extracellular Gene |
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