基于双通道结构光照明超分辨成像技术的细胞质膜脂筏研究

The cell plasma membrane model, based on lipid rafts assumption, assumed that plasma membrane co-exist phase separation of liquid disorder and liquid ordered phase. And lipid rafts play a key role in a huge range of cellular processes. Because of the resolution diffraction limit of the traditional fluorescence microscopy and small size of the lipid rafts, one cannot see lipid rafts clearly. Recent developed PALM and STED super resolution imaging technology has been used in imaging the lipid rafts. But it only detected the clusters of the lipid rafts, and cannot detect the physical environment of the lipid rafts. Moreover, environment-sensitive probe can measure the change of the liquid order phase and liquid disordered phase, which reflects the physical environment of the lipid rafts. However, the probe was only applied to the imaging system with diffraction limit, such as confocal microscopy and two-photon microscopy. This project will combine structured illumination super resolution microscopy with the environment-sensitive probe, to directly measure the heterogeneity of lipid rafts in cell membrane at sub–diffraction-limited scale, which will deepen our understanding on the structure and function of lipid rafts. And it will provide research foundation for many diseases caused by the abnormality of the structure and function of the lipid rafts, such as Alzheimer and Parkinson.

基于脂筏假设的细胞质膜模型，认为质膜同时存在液态无序相与液态有序相两种相的相分离。脂筏被认为在大量的细胞过程中起关键的作用。由于传统荧光显微镜的分辨率存在衍射极限，而脂筏尺寸又非常小，故无法真正看清楚脂筏。因此近年发展的PALM与STED超分辨成像技术已用于脂筏成像，但其只是探测了脂筏的簇，未能探测定义脂筏的物理环境。而环境敏感探针，可以测量细胞膜的液态有序相与液态无序相的变化，从而反映脂筏的物理环境。不过，此探针目前还只应用于存在衍射极限的成像系统，如共聚焦显微镜与双光子显微镜。本项目结合结构光照明超分辨显微术与环境敏感探针，在亚衍射分辨率尺度直接测量细胞膜上脂筏的多相性，从而加深对脂筏结对脂筏结构与功能的认识，为脂筏结构和功能的异常可能导致的许多疾病，如阿尔茨海默症、帕金森症等疾病提供研究基础。

肿瘤细胞的发展、迁移和入侵的过程都伴随着细胞质膜特性的改变，而脂筏是细胞质膜上富含胆固醇和鞘脂类的、动态的、不均匀的微结构域，在细胞的信号传导和运输中起着重要的作用。因此，研究不同肿瘤细胞与其质膜脂筏特性之间的关系具有重要意义，为脂筏结构和功能的异常可能导致的许多疾病提供研究基础。使用环境敏感探针di-4-ANEPPDHQ研究三种乳腺癌细胞MCF-10A、MCF-7和MDA-MB-231的迁移能力和质膜广义极化（GP）值之间的关系，结果显示迁移能力越强的细胞，其质膜的GP值越高。再通过胆固醇损耗试剂甲基-β-环糊精（MβCD）损耗胆固醇，研究胆固醇对三种细胞迁移能力的影响。实验表明胆固醇含量较高的乳腺癌细胞MDA-MB-231和MCF-7对胆固醇消耗试剂更敏感，而正常上皮细胞则对其具有一定的抵抗性。并且，上述结果也通过了其细胞迁移标志蛋白MMP-9和uPAR的基因表达水平的检测得到了验证。研究结果有助于了解脂筏微区在癌细胞发展中发挥的作用，对癌症的预防和提前诊断具有重要的意义。