近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定

doi:10.16288/j.yczz.16-017

遗传 ›› 2016, Vol. 38 ›› Issue (5): 427-435.doi: 10.16288/j.yczz.16-017

近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定

李泰明¹, 蓝文俊¹, 黄灿³, 张春², 刘晓玫²

1. 中国药科大学生命科学与技术学院,南京 210009;
2. 中国科学院苏州生物医学工程技术研究所,苏州市分子诊断和治疗技术重点实验室,苏州 215163;
3. 上海大学生命科学学院,上海 200436

收稿日期:2016-01-13 修回日期:2016-02-29 出版日期:2016-05-20 发布日期:2016-04-05
通讯作者: 刘晓玫,博士,助理研究员,研究方向：基因治疗。E-mail: lxmdov@126.com E-mail:taimingli@163.com
作者简介:李泰明,博士,副教授,研究方向：活性肽、疫苗等基因工程药物的研究。E-mail: taimingli@163.com

Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines

Taiming Li¹, Wenjun Lan¹, Can Huang³, Chun Zhang², Xiaomei Liu²

1. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;
2. Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163,China;
3. School of Life Science, Shanghai University, Shanghai 200436, China

Received:2016-01-13 Revised:2016-02-29 Online:2016-05-20 Published:2016-04-05

摘要/Abstract

摘要： 外泌体(Exosomes)是一种大小为30~100 nm的细胞外膜囊泡,与细胞的生物学功能及细胞间的信号传递有着密切的关系,尤其在癌症的诊断及治疗等领域发挥重要作用。为将外泌体更好地应用于乳腺癌肿瘤传递机制的研究,本文首先通过分子克隆手段将近红外荧光蛋白iRFP682基因和外泌体标记蛋白CD63基因克隆到含腺相关病毒(Adeno-associated virus,AAV)末端倒置重复序列(Inverted repeat terminal,ITR)的质粒载体上,构建融合表达近红外荧光蛋白和CD63蛋白的重组真核表达载体。然后再与辅助质粒共转染AAV-293细胞,包装重组腺相关病毒、纯化测量滴度后用于感染乳腺癌细胞,最后通过荧光筛选出稳定表达近红外荧光蛋白的乳腺癌细胞株。通过对乳腺癌稳定株的分离、纯化及鉴定,最终得到一个新型生物标记物：iRFP682标记的乳腺癌细胞来源的外泌体,为后续研究外泌体在乳腺癌肿瘤微环境中的分布及信号传递提供保障。

关键词: 外泌体, CD63, 近红外荧光, 荧光分子标记, 乳腺癌

Abstract: Exosomes, a population of extracellular membrane vesicles of 30-100 nm in diameter, play important roles in cell biological functions, intercellular signal transduction and especially in cancer diagnosis and therapy. To better apply exosomes in mechanistic study of breast cancer signal transduction, we constructed recombinant eukaryotic expression vector expressing the near-infrared fluorescence protein and CD63 fusion protein through cloning iRFP682 gene and exosomal marker protein CD63 gene into plasmid containing the ITR of AAV. The constructed plasmids were co-transfected with helper plasmid in AAV-293 cell lines and were packaged into rAAV. After titer measurement, the recombinant plasmids were transfected into breast cancer cell lines. The cell lines that stably expressing near-infrared fluorescence protein were selected by fluorescence. Through isolation, purification and identification, we finally obtained a new biomarker: iRFP682 labeled exosomes secreted by breast cancer cell lines, which could be used in further studies of the distribution and signal transduction of exosomes in breast cancer microenvironment.

Key words: exosomes, CD63, near-infrared fluorescent protein, fluorescent protein labeling, breast cancer

李泰明, 蓝文俊, 黄灿, 张春, 刘晓玫. 近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定[J]. 遗传, 2016, 38(5): 427-435.

Taiming Li, Wenjun Lan, Can Huang, Chun Zhang, Xiaomei Liu. Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines[J]. HEREDITAS(Beijing), 2016, 38(5): 427-435.

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近红外荧光蛋白标记乳腺癌细胞外泌体的构建及鉴定

Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines

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