单细胞功能蛋白质组学微流控芯片新方法研究

Investigation on life at single cell level has become the new challenge for bioanalytical chemistry in recent years. Currently, single cell proteomics is manly achieved by two thoroughly different strategies such as chemical cytometry and flow cytometry. In this project, we demonstrate a novel method to realize single cell functional proteomics. In brief, on a microfluidic chip cell proteins are functionally identified and labeled by fluorescently-tagged antibodies (idea learned from flow cytometry strategy). The labeled cells are then transported and selected for subsequent on-line single cell lyses, electrophoretic separation and highly sensitive laser-induced fluorescence detection (idea learned from chemical cytometry strategy). This new method not only overcomes problems in proteins identification and peak capacity of chemical cytometry, but also overcomes problems in the use of multi-color antibodies and high cost on primary cells of flow cytometry. With this novel roadmap, we will focus our interest in liver cancer, a tumor disease of the highest risk in Chinese population, and try to characterize the expressions of cancer-related signal transduction proteome, and map out the network of liver cancer signal regulations as well. This method paves a new avenue to early cancer detection and personalized medicine, and will be beneficial for fundamental researches including analytical chemistry on life science, protein science and tumor biology. We hope that it will greatly promote the development of Chinese analytical chemistry or other related disciplines and young talents training.

从单个细胞水平探讨生命过程已成为近年来生命分析化学新的挑战和机遇。目前，国际单细胞蛋白质组学主要采用化学细胞术或流式细胞术两条截然不同路线。本项目提出建立一种单细胞功能蛋白质组学新方法，以微流控芯片为平台，采用流式细胞术中蛋白质功能性标记策略，利用单色荧光抗体集对单细胞功能蛋白质组或群进行识别和标记，并进一步采用化学细胞术策略，在微流控芯片上完成细胞的运送、定位、溶胞和蛋白质分子电泳分离与激光诱导荧光检测，从而克服了化学细胞术在蛋白质鉴定上的困扰及峰容量问题，也解决了流式细胞术多色复杂标记的难点及大量原代细胞消耗的困境；采用上述新思路，重点针对中国人群发病率最高的癌症肝癌，研究单个细胞水平癌症信号相关蛋白组的差异表达，并描画出肝癌信号调控网络。本课题对于生命分析化学、蛋白质科学和癌症等基础研究以及个体化医疗等应用领域具有重要意义，并促进分析化学及其相关交叉学科的发展和人才培养。

在单个细胞水平揭示细胞异质性，是当前生命科学领域的研究的重点之一，也是生命分析化学面临的巨大挑战和机遇。本项目在国际上率先提出并建立了单细胞化学蛋白质组学新技术新方法，揭示具有重要功能的蛋白质家族在单细胞水平的表达异质性。从动物或培养细胞系获取的细胞群，通过特定设计的活性小分子探针标记后，用微流控芯片铺展成为单细胞阵列，并进一步将单个细胞进样到毛细管中裂解，释放的蛋白质采用毛细管电泳高效分离和激光诱导荧光高灵敏检测，从而克服了化学细胞术在蛋白质鉴定上的困扰及峰容量问题，也解决了流式细胞术多色复杂标记的难点及大量原代细胞消耗的困境。针对重要膜蛋白GPCR家族III，定量解析了小鼠颗粒神经元单细胞水平GABAB1a的表达差异；针对重要的细胞内功能蛋白质半胱氨酸组织蛋白酶家族，鉴别出巨噬瘤单细胞水平在该蛋白质家族不同的两种表达模式或亚型；进一步，建立了两种高通量单细胞阵列分析新方法，即液滴微点阵芯片和微通道阵列芯片，处理细胞的通量都在10000个水平。通过上述研究，在Angew Chem, Adv Mater, Anal Chem, Lab Chip等学术期刊上发表SCI学术论文16篇，其中IF>10的2篇，IF>5的6篇，IF>3的7篇，获得国家发明专利2项，培养博士4名。本项目建立的分析化学新方法，在肿瘤生物学、神经生物学和发育生物学等领域具有广泛的应用前景。