高通量单细胞分析平台研究细胞表面胆固醇与脂分子的相互作用

Interactions of the membrane cholesterol and various lipid molecules are important factors in determining cholesterol activity. The key to understanding the activity of cholesterol in the cell membrane is a comprehensive analysis of a variety of membrane lipids (especially at single-cell level), and an evaluation of the effects of the lipid proportion on cholesterol activity. Due to the importance of simultaneously detection of various lipids on a single cell surface, on the basis of last-three-year research, the project will develop a single-cell ECL imaging platform, studying the reaction of the specific proteins and membrane lipids, which produces detectable hydrogen peroxide. Optical signals will be recorded by the CCD. It is label-free single cell detection with high sensitivity and high throughput. A variety of lipids in turn react with the corresponding enzyme, so that those associated membrane lipids is detected one by one, providing information about the composition of various lipids within cell membrane. A cellular level physical model will be established to study the interactions of lipids and cholesterol activity, for the interpretation of physical and biological reasons of cholesterol activity difference. Finally, the comparison of primary disease cells with cultured cells will reveal the molecular difference in lipid interactions. For the pathological study of lipid-related diseases, the project will provide effective technology platform and biological information.

细胞膜内胆固醇和各种脂分子的相互作用是决定胆固醇活性的重要因素。理解胆固醇在细胞膜内的活性，关键在于完整分析细胞（特别在单细胞层面）膜内的各种脂的含量，评估脂的相对含量变化对胆固醇活性的影响。鉴于在单细胞表面进行多种脂的联合检测的重要性，本项目将在过去三年的研究基础上，发展单细胞电致化学发光成像研究平台，借助特异性的蛋白与细胞表面脂分子发生反应，产生可检测的双氧水；利用CCD对各细胞产生的光信号同时记录，实现高灵敏和高通量的无标记单细胞检测；通过多种脂分子与对应的酶依次发生反应，可对一个细胞膜中相关联的多种脂分子进行逐一检测，全面获得各种脂在细胞膜内的组成比例，从而在细胞层面建立脂分子相互作用和胆固醇活性的物理模型，诠释单细胞胆固醇活性差异的物理和生物学根源。最后，比较具有疾病特征的原代细胞和实验室培养细胞在脂分子相互作用方面的差异，为研究脂相关类疾病的病理提供有效的技术平台和生物学信息。

细胞膜内胆固醇和各种脂分子的相互作用密切影响细胞膜内胆固醇的活性，并进一步与脂紊乱疾病相关。本项目通过电极表面属性和结构优化进一步提高电致化学发光的信号强度，利用成像方法对单细胞表面分子进行检测，实现高灵敏、高通量的免标记单细胞分析平台。利用构建平台，对单个细胞表面含有的低含量磷脂酰丝氨酸和鞘磷脂等多种脂分子含量，及胆固醇活性进行原位检测。通过对细胞生理过程的调节，系统改变细胞表面各种脂分子（包含胆固醇）的含量，通过分析脂分子相对比例和胆固醇活性，建立脂分子相互作用与胆固醇活性的模型。最终，选取具有动脉硬化疾病、帕金森症疾病和尼曼-匹克病（又称鞘磷脂沉积病）特征的原代细胞，研究胆固醇升高、胆固醇降低和鞘磷脂升高三种情况下的脂分子相互作用对胆固醇活性的影响情况，实现了对具有脂紊乱特征原代细胞中脂分子的相互作用的系统研究。本项目为研究脂相关类疾病的病理提供了有效的技术平台和生物学信息。