基于QCM传感器实时监测水的跨膜转运研究细胞容积调节

Cell volume regulation plays an important role in various physiological processes, and its dysfunction may result in many diseases. Water transportation across plasma membrane is the main pathway for rapid cell volume regulation. However, it is infeasible to accurately detect the dynamic change of cellular mass in the process of water transportation, and to character cell volume regulation by using traditional techniques which usually suffer from poor accuracy, low sensitivity and end-point detection, etc. In our preliminary study, it is found that Quartz crystal microbalance (QCM), a kind of nanogram level of mass sensing and on-line analysis technique, can be used as an excellent tool for tracking cell volume regulation. Thus, this project firstly proposed to develop a novel analytical platform based on QCM to detect the cell volume regulation. So QCM sensor should be constructed to real-time monitoring dynamic change of cellular mass during water transportation across plasma membrane, and the new method is established for assay of cell volume regulation at high accuracy and time resolution. To evaluate the ability of cell volume regulation for different cancer cells, regulatory cell volume decrease (RVD) is used as reseach model to examine the dynamic variation in cell volume (or mass) by QCM sensor under regulating the osmotic pressure, and inhibiting of chloride channel and aquaporin 1, separately. The permeability of plasma menbrane is explored by QCM sensor monitoring water transportation and integrating with AFM and SEM imaging on nanostructure of plasma membrane. The strategy will provide a novel technique platform for relevance analysis of cell function and water, which would be applied to study physiology and pathology of diseases.

细胞容积调节参与细胞多种生理功能,其调节紊乱会引发多种疾病。水跨细胞膜转运是实现细胞容积快速调节的重要途径,然而,现有技术难以精确测量跨膜转运水质量动态变化表征细胞容积调节。前期研究发现,石英晶体微天平(QCM),一种纳克级质量型在线分析技术,可解决现有技术准确度差、灵敏度低、终点式测量细胞容积等难题。因此,本项目首次提出运用QCM发展新的分析平台研究细胞容积调节,构建QCM传感器实时监测水跨膜转运质量动态变化,建立高灵敏度高时间分辨测量细胞容积调节新方法；以低渗刺激发生调节性细胞容积减小(RVD)为模型,研究分别调控渗透压、容积敏感性氯通道和水通道蛋白1对癌细胞RVD功能的影响,分析几种癌细胞RVD功能差异；探讨RVD过程水跨膜转运与细胞膜通透性关系；建立可实时监测、精准测量细胞容积调节功能新方法。该研究将为细胞生理功能与水关联研究提供新的技术平台，对生理病理研究具有重要意义和应用前景。

细胞容积调节是细胞维持稳态和生存的重要功能，它参与细胞代谢、增殖、分化、迁移、凋亡等各种生理过程。在生理和病理条件下，细胞常遭受渗透压波动而发生细胞容积调节，其调节功能紊乱与疾病密切相关。为了维持细胞内稳态和细胞功能，细胞会启动容积调节机制，通过质膜上的通道蛋白快速转运无机离子和有机渗透物质，同时触发水分子经水通道跨膜转运。目前对于细胞容积调节的监测缺乏有效的技术方法，它需要对活细胞进行高灵敏、高精度、原位、动态分析，然而现有技术难以满足这个要求。石英晶体微天平(QCM)具有高灵敏的质量传感和免标记原位实时分析的优点。因此，本项目首次提出了以石英晶体微天平作为主要技术发展新型的分析平台用于监测细胞容积调节和水分子跨细胞膜转运，并进一步运用该分析平台成功开展了各种应用研究，包括:1)基于水分子跨膜转运和细胞容积调节建立对细胞生物功能研究的新方法；2）基于有机小分子跨细胞膜转运和细胞容积调节建立分析新方法。3)各种离子通道、水通道参与细胞容积调节机制，基于细胞体积调节和QCM技术的结合构建新型的QCM-RVD传感平台应用于离子通道（氯通道、钾通道、VRAC、TRPC）、水通道（AQP3，AQP9）以及其它有机小分子通道(尿素通道UTs)的研究，展现了全新的理念和技术方法。4）评估细胞膜通道蛋白的表达水平和活性功能，为疾病病理机制研究和诊断及预后的研究提供了新的实验证据和新的技术手段。5）发展了电化学发光生物传感器、红外光谱技术在该研究领域的应用，取得了较好的研究结果，也拓展了这些技术的应用范围。总之，在项目基金的资助下，本项目研究取得预期结果，我们构建的技术平台与方法将在生命分析科学领域中具有广阔的应用前景。