整合素avβ3受体对比超声靶向造影评价喉癌血管生成的实验研究

Angiogenesis, the recruitment of new blood vessels, occurs at a very early stage during the development and growth of different solid tumors. Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quanti?cation of tumor angiogenesis at the molecular level. Our preliminary studies have shown that the important structures of the larynx could be well visualized by high-frequency ultrasonography. Ultrasonography can provide adequate information with the location, size, intra- and extralaryngeal tissues extension, and laryngeal cartilages involvements of laryngeal carcinoma. In this project, we will combine the advantages of ultrasound with the advantages of molecular imaging through use of contrast-enhanced microbubbles targeted to Integrin avβ3, which has been shown to be over expressed in laryngeal carcinoma. In Aim 1 of this proposal, we will identify the Integrin avβ3-targeted microbubbles have a high specificity for binding to avβ3 expressing cells of laryngeal carcinoma. In Aim 2 we will test the accuracy of this targeted ultrasound imaging approach that allows detection of molecular alterations in tumor angiogenesis at different stage in subcutaneous cancer xenografts in mouse model. Molecular ultrasound imaging data will be quantitatively correlated with the expression of VEGF and micro vessel intensity obtained by immunohistochemical stains. The relationship between the parameters of time-intensity curve and the tumor size, status of lymph node, and metastasis will be also analyzed. In conclusion, the primary objective of this proposal is to identify and validate targeted microbubble contrast enhanced ultrasound for non-invasive, quantitative and objective evaluation tumor angiogenesis of laryngeal carcinoma. The successful completion of our aims will have the potential benefit to improve outcomes for patients with laryngeal cancer, and selecting and monitoring the response to anti-angiogenic therapy of laryngeal cancer.

血管生成是各种肿瘤赖以生存和侵袭的前提条件。发展新的、能准确无创并且可重复地评估肿瘤新生血管、预测肿瘤转移预后的影像技术手段，成为现代医学影像学的一个重要课题。我们的前期研究已经证实超声可清晰显示喉内的重要结抅，准确判断喉癌的发生位置、大小，可提供肿瘤的粘膜下、喉旁间隙及喉软骨侵犯等重要信息。本研究将以肿瘤新生血管内皮细胞特异性靶分子整合素avβ3为靶标制备靶向超声造影微泡，建立裸鼠人喉癌移植瘤动物模型，采用实时超声造影及时间-强度曲线定量分析技术评价该靶向超声造影剂显示喉癌肿瘤血流灌注特征的能力和效果，结合免疫组化检查方法探讨造影增强时间-强度曲线定量参数与肿瘤体积、VEGF表达、微血管密度的相关性，分析其与淋巴结转移及远处转移的相关关系，并进一步为以肿瘤血管生长因子内皮细胞为靶标的肿瘤分子成像研究及抗肿瘤血管生成的免疫和基因治疗奠定实验基础。

血管生成是各种肿瘤赖以生存和侵袭的前提条件。靶向超声分子成像技术被认为是在分子水平量化评价肿瘤血管生成的重要手段。本研究以肿瘤新生血管内皮细胞特异性靶分子整合素ανβ3为靶标制备新型靶向微泡，探讨其在体外与小鼠血管内皮细胞（ bEnd.3）靶向黏附效果。进一步地，课题组将RGD靶向微泡用于喉癌移植瘤超声分子成像实验评价整合素 ανβ3在不同生长阶段肿瘤新生血管中表达的特点。 结果表明：1）制备获得的靶向微泡呈球型，具有良好的分散性。靶向微泡平均粒径大小为2.36 ± 1.7 μm，粒径分布主要位于1 ~ 10 μm，微泡主峰位于 ～1.68 μm，而在～0.6 μm, ～3.99 μm 和 ～6.85 μm均有较小的次峰存在。2）RGD靶向微泡组与空白对照组比较，RGD靶向微泡组中微泡黏附个数显著性增多，单个视野微泡黏附个数及单个细胞表面黏附微泡个数随着RGD微泡浓度的提高逐渐增加。而αvβ3阴性表达MCF-7细胞表面仅有少量RGD靶向微泡黏附。采用抗-alpha(v) 抗体预处理封闭细胞表面整合素受体ανβ3后，抗体封闭组黏附在单个视野微泡个数及单个细胞表面微泡个数均显著降低，并随着抗-alpha(v) 抗体浓度增高而下降。表明RGD靶向微泡与血管内皮细胞是由RGD肽与整合素受体介导的特异性结合。3）RGD-MBs于细胞表面靶向结合速率因剪切应力的大小不同而改变。低剪切应力条件下靶向微泡与细胞黏附率较低，随着剪切应力的增大，黏附率随之增大。剪切应力为1.5 dyne/cm2时，微泡与细胞表面黏附率最大，达到最大峰值（48.72 ± 4.26）个/min。4）体内超声分子显像实验结果显示RGD靶向微泡可有效评价整合素ανβ3在肿瘤血管新生中的表达，随着肿瘤体积增大，喉癌中靶向微泡超声信号强度及ανβ3表达水平呈逐渐降低的趋势。5) 喉癌超声造影表现为：a. 肿块内部不均匀整体性高增强为主，b.造影剂向肿块的外缘及周围明显浸润，肿块周围见造影剂通过血管进入，c. 造影时间-强度曲线特征表现为增强开始时间、达峰时间均较早。结论：超声分子显像可有效评价整合素ανβ3在肿瘤血管新生中的表达，对于喉癌的早期诊断及预后监测具有重要价值。