基于微流控芯片技术的单细胞凋亡动力学过程与基因表达分析研究

Single cell is the fundamental building brick of life. However, the majority understanding of cellular biology and genetics has been collected through the bulk experiment from large population of cells. Traditional methods for single cell analysis have been limited by the cost and throughput required to pick up wanted individual cells from large population and the difficulties coupled with analyzing small amounts of starting material. One of the hallmarks among the single cell driven phenomenon is apoptosis, which refers to a specific form of programmed cell death, takes place in tissues to guarantee the welfare of the whole organism though the elimination of unwanted cells. Dysfunction in apoptosis can lead to disease states including cancer. The techniques for detection apoptotic cell are therefore highly interesting. Notwithstanding, nowadays techniques for probing apoptosis are based on bulk cell analysis and not suitable for single cell level analysis or incapable of real-time monitoring for understanding the heterogeneity during apoptosis. Few methods, such as flow cytometry and fluorescent microcopy are incapable of accessing the genomic data of the single cells. Microfluidic approach can be used to plumb single cell issues with several promising advantages such as scale compatibility of mammalian cells, friendly coupling with imaging process, flexibility of manipulating cells and automation. Aiming on the dynamic analysis of single apoptotic cells and the gene expression study by using microfluidic device, our project is a highly interdisciplinary in research scope. The basic technology of our research is the multiple soft lithography which can be used to fabricate microfluidic device. By integrating different micro-scale tools such as micro-valve, micro-well and micro-dam structures, we can manipulate the single tumor cells in a device with precisely controlling of the microenvironment. In addition, apoptotic cells will be monitored by high-throughput imaging based on specific fluorescent staining to investigate the dynamic process during programmed cell death. More over, the apoptotic cells that may particularly be interested in will be further analyzed to assess its gene expression patterns by delivering to downstream component or transferring to outside of the chip. Thus, the full analysis of single cell programmed death will be realized by integration of acquired data from dynamic imaging and genome information. The accomplishment of this project is not only a innovation of microfluidic device but also a paradigm of quantitatively study of single cells from a microsystem.

本项目面向单细胞凋亡分析和研究需求，发展适合于单细胞分析的微流控芯片实验室技术。利用多层软刻蚀技术以PDMS 为材料制备集成的微米级细胞培养体系，让单个及少数细胞在可精密调控的培养微腔中生长。利用多层软蚀刻方法加工整合微阀、微坑阵列和微围堰结构等微结构的大规模集成，精确操控单个细胞的凋亡受激并控制外界诱导因子作用的时间和强度，结合单细胞显微成像自动分析系统，在更精确可控的尺度上研究高通量单细胞的受激凋亡响应和进行实时动态观察。同时，基于微流芯片的规模集成和灵活可操作性，完成单细胞在凋亡之后的操控、转运和对凋亡调控的多个关键基因等生理生化指标分析，实现对单细胞凋亡的完全分析研究。最后，针对单细胞高通量分析的特点，对该系统中的研究与传统的群体细胞凋亡研究方法做比较，以期能够对单细胞的凋亡分析有更加深入的理解，对单细胞在凋亡过程中的异质性做出更加深刻的阐释。

本项目针对单细胞生物学，致力于发展出适合与单细胞分析的微流控技术。2014年，在《美国科学院院刊》（PNAS）上首次报道了利用多层微流控芯片技术加工用于单个细胞全转录组测序的微流控芯片。该研究应用微流控芯片装置实现了单个细胞的高质量测序样品的准备，极大地提高了单细胞全转录组测序分析结果的准确性与可靠性。2015年，在Anal. Chem. 上首次报道了基于微流控芯片细胞图案化技术和荧光定量PCR技术联用，可以在一块芯片上进行多种细胞的成像分析和基因表达分析。对药物耐药性的筛选试验分析过程仅需要小于400个细胞。利用单细胞微坑阵列进行了单细胞凋亡动力学的分析，单细胞的阵列密度大于2000个／mm2，能够进行单细胞凋亡的动力学分析，是目前密度最高的单细胞阵列进行凋亡动力学的研究报道，取得了较大的进展。目前正在组织进行稿件的撰写。