遗传 ›› 2017, Vol. 39 ›› Issue (1): 66-74.doi: 10.16288/j.yczz.16-217

• 技术与方法 • 上一篇    下一篇

肺癌患者外周血中循环肿瘤细胞的快速分离与体外培养

吴文君(),王智华,王卓,邓宇亮,施奇惠()   

  1. 上海交通大学系统生物医学研究院,上海200240
  • 收稿日期:2016-06-17 修回日期:2016-10-19 出版日期:2017-01-20 发布日期:2017-12-24
  • 作者简介:吴文君,硕士研究生,专业方向：循环肿瘤细胞快速分离与体外培养。E-mail: 1126958030@qq.com|施奇惠，教授，研究方向：微流控芯片技术的单细胞分析及临床诊断芯片。E-mail: qihuishi@sjtu.edu.cn
  • 基金资助:
    国家自然科学基金项目(81371712,81501613);上海市自然科学基金项目(13ZR1456100);高等学校博士学科点专项科研基金项目(20130073120106)

Fast isolation and ex vivo culture of circulating tumor cells from the peripheral blood of lung cancer patients

Wenjun Wu(),Zhihua Wang,Zhuo Wang,Yuliang Deng,Qihui Shi()   

  1. Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai 200240, China
  • Received:2016-06-17 Revised:2016-10-19 Online:2017-01-20 Published:2017-12-24
  • Supported by:
    the National Natural Science Foundation of China(81371712,81501613);the Natural Science Foundation of Shanghai(13ZR1456100);Research Fund for the Docoral Program of High Education of China(20130073120106)

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摘要：

循环肿瘤细胞(circulating tumor cells, CTCs)是从肿瘤病灶脱落并进入外周血液循环的处于游离状态的肿瘤细胞,代表了肿瘤病灶的分子特征,可用于对肿瘤的“液体活检”。但外周血中CTCs数目极为稀少,使得后续针对CTCs的分子与功能分析面临巨大挑战。鉴于此,本文建立了一种基于微流控芯片和免疫磁珠的能够快速从肺癌患者的外周血中分离CTCs的方法。该方法直接针对全血进行一步分离,可避免血液样本预处理及富集等过程对细胞造成的损伤,从而有效地保护CTCs的活性(>90%)。分离得到的CTCs可富集在小体积中(80 μL),实现高密度的细胞培养,完成体外扩增,扩增后的CTCs可以被进一步冻存、复苏及再次增殖培养,表明已经对患者血液中的CTCs成功建系。本文进一步对CTCs进行了基因突变(EGFR、KRAS、PIK3CA、TP53和BRAF)检测及荧光标记葡萄糖类似物(2-NBDG)摄取的功能分析,证明CTCs存在较大异质性。本研究成功实现了对外周血中稀少的CTCs进行体外培养,并对CTCs进行了基因、蛋白、功能等各个层面的分析,这对于肿瘤精准医疗具有重要的临床意义。

关键词: 循环肿瘤细胞, 微流控芯片, 体外培养, 液体活检

Abstract:

Circulating tumor cells (CTCs) are free tumor cells shed from tumor site and enter into blood circulation. CTCs represent a reliable source of tumor cells for the molecular characteristics of the original tumor. However, the extraordinary rarity of CTCs makes the subsequent molecular and functional analysis technically challenging. Here, we describe a one-step microfludics-based immunomagnetic isolation method to isolate CTCs directly from the whole blood of lung adenocarcinoma patients. This method avoids harsh sample preparation and enrichment steps, and therefore preserves the viability (>90%) of CTCs during the in vitro isolation. The isolated CTCs are enriched in small volume (80 μL) and cultured ex vivo that leads to successful ex vivo expansion. The expanded CTCs can be frozen and thawed, which shows cell line property. Genetic sequencing on EGFR、KRAS、PIK3CA、TP53 and BRAF and metabolic assay (2-NBDG) are utilized to characterize the expanded CTCs. Our results demostrated that this method is suitable for ex vivo expansion of CTCs facilitates. The genomic, proteomic and metabolic analyses of CTCs have guiding significance in tumor precise treatment.

Key words: circulating tumor cells, microfluidics, ex vivo culture, liquid biospy