微气泡稳态声空化微流场对细胞膜通透性和细胞骨架的作用机制研究

Gene therapy is one of the most promising therapeutic strategies for cancer treatment. Despite increasing interesting in the non-invasive US-assisted gene/drug delivery to tumor site, the mechanisms involved in the related processes have not been well known. The hypothesis of the current project is that the acoustic microstreaming induced by stable cavitation bubble would play an important role in the microbubble-mediated gene/drug delivery. The proposed studies are: (1) improving the theoretical modeling of cavitation bubble-induced microstreaming with considering microbubble shell properties, the constrained effect of vessels and the non-Newtonian blood flow environment, and investigating the dependence of microstreaming pattern on ultrasound parameters based on Finite Element Method simulations; (2) setting up a high-speed micro-PIV system to precisely measure the acoustic microstreaming field induced by stable cavitation bubble, and make a comparison between the experimental and theoretical results; (3) simultaneously monitoring the effect of microbubble-induced acoustic microstreaming on cell membrane and cytoskeleton based on GFP protein tagging and PI dying technologies; and (4) under varied experimental conditions, investigating the correlation between microstreaming-induced shear stress, membrane sonoporation, cytoskeleton disassembly and cell viability. The results of proposed studies will give us better understanding on the effect of microstreaming induced by stable cavitation bubble on the cell membrane and cytoskeleton, which is important for optimizing and controlling the microbubble-enhanced drug delivery for cancer treatment in clinic.

基因疗法是目前生物医学领域研究热点，而超声介导基因/药物转染以其生物毒性小、设备简单、易于时空定位、可实现无损治疗等优点日益得到多方关注，但其相关作用机制研究仍不完善。本课题拟在考虑微泡包膜特性、血管局限作用及非牛顿流体血液环境基础上，完善微泡声微流场理论建模工作，并基于数值仿真研究超声参数对其结构模式的影响；构建高速micro-PIV实验系统来实时精确测量稳态空化泡激发的声微流场，并与理论模型相验证；此外，利用荧光标记和荧光染色技术分别表征HeLa细胞骨架和细胞膜，通过实时测量其荧光强度变化来研究微泡稳态声空化行为对细胞膜和细胞骨架的作用；同时，研究不同参数条件下，声微流场激发的剪切应力、细胞膜声穿孔、细胞骨架解聚及细胞活性之间的相关性。在此基础上，深入研究微泡稳态声空化效应对细胞膜和细胞骨架的作用机制、探索提高超声转染效率并保持细胞活性的优化参数范围，从而更有效地控制和优化临床疗效。

基因疗法是目前生物医学领域研究热点，而超声介导基因/药物转染以其生物毒性小、设备简单、易于时空定位、可实现无损治疗等优点日益得到多方关注，但其相关作用机制研究仍不完善。本项目的研究内容主要包括：1. 考虑微气泡包膜层物理特性（如弹性系数、粘度或厚度等）、血管局限作用及血液的非牛顿流体环境等因素，进一步完善微泡声微流场的理论建模工作，构建了非线性包膜粘弹模型（NSEV）；通过数值仿真研究不同参数对刚性壁面附近微泡声微流场的影响；2. 构建了高速micro-PIV实验观测系统，实现了对微泡稳态声空化微流场的精确实时观测；3. 基于荧光标记技术构建细胞骨架可稳定表达绿色荧光的HeLa细胞株，并利用PI红色荧光染料表征细胞膜声穿孔情况。通过量化观察红、绿两种荧光强度随时间演化行为，研究微泡稳态声空化效应对细胞膜和细胞骨架的作用；4. 在不同参数条件下（如超声驱动声压、作用时间、造影剂微泡浓度及细胞与微泡之间距离等），实时测量并研究细胞膜通透性变化与细胞骨架解聚程度之间的相关性。本项目研究结果对于深入了解微泡稳态声空化效应对细胞膜和细胞骨架的作用机制、探索提高超声转染效率并保持细胞活性的优化参数范围具有重要科学意义，从而更有效地控制和优化临床疗效。本项目在研究过程中按照研究计划实行，迄今为止，课题组已经发表了SCI文章21篇文章；申请专利1项；培养博士生1名，硕士生5名；在国际学术会议做口头报告7次，邀请8位外国专家访华，超额完成了预期研究目标。