肺癌循环肿瘤细胞的单细胞<em>EGFR</em>基因突变检测

孙帅, 邓宇亮

doi:10.16288/j.yczz.15-130

遗传 >

2015 , Vol. 37 >Issue 12: 1251 - 1257

DOI: https://doi.org/10.16288/j.yczz.15-130

技术与方法

肺癌循环肿瘤细胞的单细胞EGFR基因突变检测

展开

上海交通大学,系统生物医学研究院,上海200240

孙帅,硕士研究生,专业方向：癌症患者血液循环肿瘤细胞的基因检测。E-mail: sunshuai_good@163.com

收稿日期: 2015-07-16

网络出版日期: 2015-09-22

基金资助

国家自然科学基金项目（编号：21205077）,上海市自然科学基金项目（编号：13ZR1456100）和教育部博士点基金;（编号：20130073120106）资助

收起

Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer

Expand

Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China

Received date: 2015-07-16

Online published: 2015-09-22

Fold

摘要

循环肿瘤细胞（Circulating tumor cells,CTCs）是从肿瘤原发病灶脱落并侵入外周血循环的肿瘤细胞。由于CTCs存在较大的异质性,其与癌症发展转移密切相关,但目前尚缺乏有效的CTCs单细胞异质性检测方法。鉴于此,本文发展了在单细胞层面对CTCs进行基因突变的检测方法并用于单个肺癌CTC的EGFR（Epidermal growth factor receptor）基因突变检测。首先用集成式微流控系统完成血液中稀有CTCs的捕获,接着将CTCs释放入含有多个微孔的微阵列芯片中,得到含有单个CTC的微孔,通过显微操作将单个CTC转入PCR管内完成单细胞基因组的放大,并进行单细胞的EGFR基因突变检测。以非小细胞肺癌细胞系A549、NCI-H1650和NCI-H1975为样本,通过芯片与毛细管修饰、引物扩增条件（复性温度、循环次数）的优化,结果显示在复性温度59℃、30个循环次数的条件下,引物扩增效果最优。利用该方法成功地对非小细胞肺癌（Non-small cell lung cancer, NSCLC）患者的血液样本进行了测试。从患者2 mL血液中获取5个CTCs,分别对其EGFR基因的第18、19、20、21外显子进行测序,发现该患者CTCs均为EGFR野生型。研究结果证明此检测方法可以灵敏地用于单个CTC基因突变的检测,在临床研究上具有重要的指导意义。

关键词： 循环肿瘤细胞; 微流控芯片; 单细胞基因突变检测; EGFR

本文引用格式

孙帅, 邓宇亮 . 肺癌循环肿瘤细胞的单细胞EGFR基因突变检测[J]. 遗传, 2015 , 37(12) : 1251 -1257 . DOI: 10.16288/j.yczz.15-130

Abstract

Circulating tumor cells (CTCs) are cells that shed from a primary tumor and enter the peripheral blood circulation. The CTCs are closely associated with tumor development and metastasis because of its high heterogeneity. However, there are still no effective methods to detect single-cell heterogeneity of the CTCs. To this end, we developed a method to detect gene mutation in CTCs at the single-cell level and applied it to the detection of EGFR gene mutation in single lung cancer CTC. Specifically, the rare CTCs were captured from blood using an integrated microfluidic system, and then were released into a microchip with thousands of nanoliter wells to isolate single CTC. The single CTC was then transferred into a PCR tube under the microscope for single-cell genome amplification and detection of EGFR gene mutation. We firstly modified chip and capillary and optimized PCR conditions (annealing temperature, number of cycles) using non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H1650 and NCI-H1975 as samples, which showed maximal amplification after 30 cycles with an annealing temperature at 59℃. We then successfully detected blood samples from NSCLC patients using this method. 5 CTCs were obtained from 2 mL patient's blood and the sequencing of EGFR exons 18, 19, 20 and 21 showed no mutations. Our results demonstrated that this method is sensitive enough to detect gene mutation in single CTC and has guiding significance in clinic research.

Key words： circulating tumor cells; microfluidic chip; single-cell gene mutation; EGFR

参考文献

[1] 李占稳. 肺癌细胞系KRAS突变检测及联合通路抑制剂的生长抑制作用[学位论文]. 北京协和医学院, 2013: 11-14.
[2] Paz-Ares L, de Marinis F, Dediu M, Thomas M, Pujol JL, Bidoli P, Molinier O, Sahoo TP, Laack E, Reck M, Corral J, Melemed S, John W, Chouaki N, ZimmermannAH, Visseren-Grul C, Gridelli C. Maintenance therapy with pemetrexed plus best supportive care versus placebo plus best supportive care after induction therapy with pemetrexed plus cisplatin for advanced non-squamous non-small-cell lung cancer (PARAMOUNT): a double-blind, phase 3, randomised controlled trial. Lancet Oncol , 2012, 13(3): 247-255.
[3] Goto K, Satouchi M, Ishii G, Nishio K, Hagiwara K, Mitsudomi T, Whiteley J, Donald E, McCormack R, Todo T. An evaluation study of EGFR mutation tests utilized for non-small-cell lung cancer in the diagnostic setting. Ann Oncol , 2012, 23(11): 2914-2919.
[4] Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura CV, Inserra E, Diederichs S, Iafrate AJ, Bell DW, Digumarthy S, Muzikansky A, Irimia D, Settleman J, Tompkins RG, Lynch TJ, Toner M, Haber DA. Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med , 2008, 359(4): 366-377.
[5] Oxnard GR, Paweletz CP, Kuang YN, Mach SL, O'Connell A, Messineo MM, Luke JJ, Butaney M, Kirschmeier P, Jackman DM, Jänne PA. Noninvasive detection of response and resistance in EGFR -mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res , 2014, 20(6): 1698-1705.
[6] Wang ZJ, Chen R, Wang SH, Zhong J, Wu MN, Zhao J, Duan JC, Zhuo ML, An TT, Wang YY, Bai H, Wang J. Quantification and dynamic monitoring of EGFR T790M in plasma cell-free DNA by digital PCR for prognosis of EGFR-TKI treatment in advanced NSCLC. PLoS?One , 2014, 9(11): e110780.
[7] 李海静, 刘岩, 郝海生, 杜卫华, 赵学明, 王栋, 秦彤, 马友记, 朱化彬. 表皮生长因子受体与肺脏发育的关系. 遗传, 2012, 34(1): 27-32.
[8] 赵宝霞. EGFR突变型NSCLC的EGFR-TKI耐药机制体外研究[学位论文]. 大连医科大学, 2013: 15-17.
[9] den Toonder J. Circulating tumor cells: the Grand Challenge. Lab Chip , 2011, 11(3): 375-377.
[10] Wang ST, Wang H, Jiao J, Chen KJ, Owens GE, Kamei K, Sun J, Sherman DJ, Behrenbruch CP, Wu H, Tseng HR. Three-dimensional nanostructured substrates toward efficient capture of circulating tumor cells. Angew Chem Int Ed , 2009, 48(47): 8970-8973.
[11] 林炳承. 微流控芯片实验室及其功能化. 中国药科大学学报, 2003, 34(1): 1-6.
[12] Sun J, Masterman-Smith MD, Graham NA, Jiao J, Mottahedeh J, Laks DR, Ohashi M, DeJesus J, Kamei K, Lee KB, Wang H, Yu ZTF, Lu YT, Hou S, Li KY, Liu M, Zhang NG, Wang ST, Angenieux B, Panosyan E, Samuels ER, Park J, Williams D, Konkankit V, Nathanson D, Van Dam RM, Phelps ME, Wu H, Liau LM, Mischel PS, Lazareff JA, Kornblum HI, Yong WH, Graeber TG, Tseng HR. A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens. Cancer Res , 2010, 70(15): 6128-6138.
[13] Qiu DM, Silva L, Tonkovich AL, Arora R. Micro-droplet formation in non-Newtonian fluid in a microchannel. Microfluid Nanofluid , 2010, 8(4): 531-548.
[14] 宋文斌, 董朝青, 任吉存. 微液滴微流控芯片: 微液滴的形成、操纵和应用. 分析科学学报, 2011, 27(1): 106-112.
[15] Eyer K, Kuhn P, Hanke C, Dittrich PS. A microchamber array for single cell isolation and analysis of intracellular biomolecules. Lab Chip , 2012, 12(4): 765-772.
[16] Umemura K, Miyabayashi T, Taira H, Suzuki A, Kumashiro Y, Okano T, Mayama S. Use of a microchamber for analysis of thermal variation of the gliding phenomenon of single Navicula pavillardii cells. Eur Biophys J , 2015, 44(3): 113-119.
[17] 廖红华, 廖宇, 易金桥, 徐建. PCR芯片中微腔室高度及加热器结构优化设计. 湖北民族学院学报(自然科学版), 2009, 27(4): 403-407.
[18] Wang MH, Zhu D. Fabrication of multiple electrodes and their application for micro-holes array in ECM. Int J Adv Manuf Technol , 2009, 41(1-2): 42-47.
[19] 张慧妍, 张珍, 吉邢虎, 何治柯. 纸基微孔阵列芯片比色法检测乳酸脱氢酶. 分析化学, 2014, 42(9): 1276-1280.
[20] Deng YL, Zhang Y, Sun S, Wang ZH, Wang MJ, Yu BQ, Czajkowsky DM, Liu BY, Li Y, Wei W, Shi QH. An integrated microf

Options

文章导航

〈

〉

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献