单细胞数字PCR技术用于肺癌循环肿瘤细胞的鉴定分析

In recent years, lung cancer has become the most common cause of cancer-related death in human beings. Overall, no more than 15% of people diagnosed with lung cancer per year survive five years after the diagnosis. The existing techniques for lung cancer diagnostics including low-dose spiral CT, sputum cytology and RT-PCR have different problems for early-stage diagnosis, which might be the main reason of high mortality rate of this desease. Herein, we propose a technique based on single-cell study of circulating tumor cell (CTC) through physical and immuno sorting and single-cell digital PCR. The proposed technology has combined the advantages of microfluidic chip and traditional molecular techniques and focused on the gene expression, mutation and heterogeneity of CTC at single cell level. The methodology mainly composed of three parts. First, CTCs are sorted and isolated from most matrix materials of sample through gradient centrifugation, immunoseparation. Then CTCs would be distinguished by immunofluorescence under multichannel fluorescent microscope. A capillary microscopy operation system would be employed to pick up individual CTCs and prepare the single cell sample. Finally we used digital PCR technique for gene profiling and mutation detection of CTCs at single cell level. Campared with most of the current work which directly analyze the blood sample for expression of cancer biomarker, our work would be more accurate and reliable. The outcome of this technology would promote the research and application of minimal invasive diagnostics for large scale screening of lung cancer. Moreover our work would provide a new platform and important information for early diagnosis and indiviaual treatment of lung cancer.

肺癌是常见的恶性肿瘤之一，现有的诊断技术难以达到早期诊断的目的，是该疾病的死亡率居高不下的重要原因。鉴于此，我们提出一种以循环肿瘤细胞（CTC）为研究对象的单细胞数字PCR基因分析技术。该技术从分子生物学角度出发，结合微流控芯片的技术优势，在单细胞水平研究肿瘤细胞的基因表达、突变情况以及CTC的异质性。该课题主要包括三部分工作，首先采用梯度离心、免疫分离和免疫荧光技术从血液样本中分离富集CTC。接着借助荧光成像技术寻找免疫荧光标记阳性的CTC以及不具有癌症标记物抗原的CTC。通过毛细管显微操作技术将单细胞逐个取出。最后，采用数字PCR技术对单细胞内肿瘤基因的表达水平和突变情况进行分析。本项目提出以血液中的CTC为研究对象，相对于直接对血液样本进行分析更加准确可靠，本项目的成果将有助于提高微创检测技术用于模癌症筛研究的准确性和实用性，为肺癌早期诊断和个体化治疗提供新的技术平台和重要信息。

目前围绕肿瘤干细胞基因分析展开的一系列研究工作已成为包括生命科学、生物医学、药物学等诸多领域关注的焦点。我们开发了一种基于微流控技术的液滴阵列芯片用于单细胞RT-qPCR检测和基因表达分析。该系统可以生成纳升甚至皮升级液滴阵列，并将细胞悬液、试剂等分步加入到每个液滴中，分别完成细胞包封、裂解、反转录和PCR扩增步骤。利用该系统，我们五分钟内生成了100个2nL体积的液滴阵列，并且实现了单个Huh-7细胞中mir-122表达分析。本项工作的意义在于建立了一种简单易用、可操纵性强的单细胞分析平台，与文献报道的其他单细胞分析技术不同的是，该系统生成的液滴是半封闭的体系，可以设计程序向反应体系中加入试剂或终止反应进行，操作灵活，适合用于多种分析体系，是一种通用的单细胞分析平台。