低强度聚焦超声协同靶向微泡抗肿瘤血管生成方法及瞬态空化力生物学效应机制

Endothelial cell apoptosis is a hot research point in antiangiogenic therapy. Based on the fact that apoptosis could be induced by low intensity with microbubbles, a potential antiangiogenic therapy with low intensity focused ultrasound (LIFU) and targeted microbubble was proposed in the project. The following research works will be completed. (1) the parameters suitable for LIFU induced apoptosis with targeted microbble would be clarified; Apoptosis signals triggered by transient cavitation will be discussed; Molecular mechanism would be illustrated by analyzing the functions of key proteins which expressed in different way because of LIFU irradiation with targeted microbubbles; Moreover, the tumor growth inhibition would be tested and verified in vivo. (2) A single cell-single bubble-LIFU-acoustic and light confocal superhigh speed/fluorescence microscopy system would be constructed with the microfluidic which mimics the physiological/ pathological blood flow conditions. The transient cavitation could be observed in controlled experimental conditions, as well as its mechano-biological effects on the endothelial cells. It would be significant to clarify the physical conditions necessary to monitor and control the interactions between targeted microbubble and cell in real time. The project is meaningful to illustrate the mechanism and to develop a novel antiangiogenic therapic protocol with LIFU and targeted microbubbles.

血管内皮细胞凋亡已成为当前抗肿瘤血管生成治疗的研究热点。在超声及微泡空化诱导细胞凋亡的基础上，本项目提出低强度聚焦超声（LIFU）协同靶向微泡抗肿瘤血管生成方法，并主要研究：（1）探明LIFU协同靶向微泡诱导血管内皮细胞凋亡的物理参数；研究瞬态空化作用下血管内皮细胞凋亡相关信号通道；筛选LIFU协同靶向微泡作用下血管内皮细胞表达差异的关键蛋白，分析其力分子生物学作用机制；并在动物模型上验证其对肿瘤生长的抑制作用。（2）在逼近生理/病理血流条件的微流芯片上集成单细胞-单微泡-LIFU-声光共焦超高速/荧光显微成像系统，观察严格控制的物理条件下LIFU 场中靶向微泡的瞬态空化现象及其对血管内皮细胞的力生物学效应，为实时监测和控制靶向微泡与细胞相互作用的物理条件提供依据。本项目的完成对阐明LIFU协同靶向微泡抗肿瘤血管生成治疗机制有重要意义，为发展抗肿瘤血管生成物理治疗方法奠定基础。

本项目的研究目标是建立低强度聚焦超声协同微泡诱导凋亡的抗肿瘤生长辅助治疗方法，并阐明其物理作用机理、生物学效应与分子生物学机制。研究主要完成的工作包括：（1）针对低强度聚焦超声协同微泡的瞬态物理机制和分子生物学效应，构建了逼近生理/病理血流条件的声光共焦低强度聚焦超声-超高速显微成像及被动空化检测系统；（2）解决单/多焦点模式下的声光共焦问题，并自主设计和制备填埋透明仿体血管模型、纤维素管模型和原位肠系膜微血管模型，讨论了微泡动力学对细胞的瞬态作用条件和机制。研究通过实验观察了多种光声空化成核和泡动力学作用模式，并结合空化成核和振动的数学模型，讨论了空化模式与温度、生物界面粘弹性、微泡尺寸和超声参数等的关系，为空化的有效控制奠定了重要基础。（3）在分子生物学效应方面，通过关键蛋白的筛选和功能分析，确定了空化效应下线粒体功能及其线粒体膜通透性膜孔（mPTP）、钙离子浓度与钙离子通道以及细胞内外活性氧相关信号通道在超声协同微泡的空化作用诱导细胞凋亡中的机制。此外，针对研究过程中国外审稿人普遍关心的驻波场分布和驻波场对细胞的影响等问题，采用声致化学方法的方法研究了细胞辐照系统中的空化分布并讨论了其对细胞活性的影响。本项目为实时监测和控制靶向微泡与细胞相互作用的物理条件提供依据，对阐明低强度超声协同微泡抗肿瘤血管生成治疗机制有重要意义，为发展抗肿瘤血管生成新物理治疗方法奠定了重要基础。