质膜去极化在声空化介导细胞凋亡中的作用机理研究

The interaction between the low intensity ultrasound pulses and microbubbles is known to generat acoustic cavitation that may mediate cell apoptosis,which offers an effective approach for clinical tumor treatment.Noninvasion，focusing on the targeted objective and low cost are the advantages of this physical treatment of disorders. When the combined use of the low intensity ultrasound and microbubbles is exposed to the cell, acoustic cavitation will firstly give rise to the change of the electrophysiological status of plasma membrane, which is the outmost protective barrier of the cell. It is no doubt that plasma membrane depolarization plays the important role in triggering the intracellular apoptotic pathways. In this research, based on the establishment of the ultrasound exposure setup and the real-time measurement of acoustic cavitation, the in vitro cancer cell line is adopted to ananlyze the interraction among the acoustic cavitation dosage, plasma membrane depolarization and intracellular cations, ion channels and plama membrane ATPase transporter, and reveal the signal conduction from the changes of these intracellular cations and membrna proteins to the intrinsic and extrinsic apoptotic pathways induced by acoustic cavitation. We expect to achieve a more complete understanding of the role of the plasma membrane depolarization during the apoptosis mediated by acoustic cavitation, and to discover the targeted proteins quickly reponse to the acoustic cavitation, and present the strategy for improving the apoptotic efficiency mediated by acoustic cavitation.These findings will supply the full and complete understanding of therapeutical potential of acoustic cavitation, and establish a solid theroretic foundation for clinical tumor therapy by the low intensity ultrasound with combination of microbubbles.

低强度超声联合微泡的声空化效应能够介导细胞凋亡为临床肿瘤治疗提供了又一选择。其无创、可靶向聚焦以及低成本的独特优点彰显了这一物理疗法的优势。在超声，微泡和细胞相互作用中，质膜作为细胞最外层的保护屏障，直接接受声空化的能量刺激，其电生理状态的变化-去极化在开启细胞内部凋亡通路中起着重要的作用。本研究在对声空化效应进行实时测量的基础上，以体外癌细胞系为模型，通过分析声空化效应剂量、质膜去极化和细胞内单价阳离子浓度的变化、质膜上相关离子通道蛋白及载体蛋白活性之间的相关关系，以揭示这些离子和膜蛋白同声空化效应介导的外源性及内源性线粒体凋亡途径之间的信号传导关系。通过该项研究，期望深入理解质膜去极化在在声空化介导细胞凋亡中的作用机理，发现能对声空化效应迅速响应的靶向蛋白，提出改进声空化介导细胞凋亡效率的策略，为声空化走向临床肿瘤治疗提供充分、可靠的理论基础。

低强度超声联合微泡的声空化效应能够介导细胞凋亡为临床肿瘤治疗提供了又一选择。其无创、可靶向聚焦以及低成本的独特优点彰显了这一物理疗法的优势。本项目在构建声空化处理和实时显微观测平台的基础上，以体外肿瘤细胞为模型，着重研究超声空化介导细胞质膜穿孔后，细胞质膜膜电势的变化特征及其在声空化介导细胞凋亡中的作用，同时分析了多样性声致穿孔的多样性特征和非声学参数之间的关系。本项目研究发现：. 1）超声空化处理之后，声致穿孔细胞的质膜电势产生去极化，且具有多样性的特征，具体为：a. 质膜穿孔但可修复的细胞显示暂时的质膜电势去极化特征；b.质膜穿孔但不可修复的细胞显示持续的质膜电势去极化特征；c.临近声致穿孔细胞的细胞（其质膜未出现穿孔）经历暂时的质膜电势去极化。. 2）超声空化处理之后，细胞内内质网动态失衡激活内源性线粒体促凋亡通路。具体表现为：a.随着处理后时间的递增，声致穿孔细胞内质网功能逐渐丧失以及线粒体膜电势持续去极化；b.内质网具有氧化蛋白折叠功能的蛋白PDI和Ero1-La表达逐渐增多，内质网跨膜应力敏感蛋白PEAK和IRE1-α被逐渐激活，表明内质网功能逐步失调；c.内质网到线粒体的促凋亡蛋白CHOP及JNK的表达增加，抑制了抗凋亡蛋白Bcl-2的表达，且Caspase-9凋亡起始蛋白被逐渐剪切，导致内源性凋亡通道开启，细胞进入凋亡程序。. 3）声致穿孔的多样性特征和非声学参数相关。a.相对较小微泡的原位破裂(直径D ＜ 5.5 μm)导致可预测的声致穿孔，其程度和微泡和细胞之间的距离（d）正相关。当d/D ＞ 1时，声致穿孔不会发生；当d/D ＜ 1时，导致可恢复性的声致穿孔发生；b.相对较大微泡(直径D ＞ 5.5 μm)在破裂前产生迁移运动，导致不可预测的声致穿孔类型。当其远离细胞迁移时，没有或者可恢复性的声致穿孔发生，而当其朝细胞迁移时，导致不同程度的可恢复性声致穿孔或不可恢复性的声致穿孔。C.较小微泡的个数和声致穿孔类型正相关。. 以上这些全新的发现有助于深入理解超声，微泡以及细胞之间相互左右的生物物理过程，初步揭示了质膜去极化在在声空化介导细胞凋亡中的作用机理，为声空化走向临床肿瘤治疗提供充分、可靠的理论基础。