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

[an error occurred while processing this directive]

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

• Research Articles • Previous Articles Next Articles

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

Abstract

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

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.

References

[1] Wu XG, Peng SB, Huang Q. Transcriptional regulation of breast cancer resistance protein. Hereditas (Beijing) , 2012, 34(12): 1529-1536. 吴新刚, 彭姝彬, 黄谦. 乳腺癌耐药蛋白基因的转录调控机制. 遗传, 2012, 34(12): 1529-1536.
[2] Yuan Y, Zhu ZY, Lai ZC, Zhang WX. Progress on breast cancer metastasis suppressor 1 (BRMS1). Hereditas(Beijing) , 2006, 28(9): 1141-1148. 袁圆, 朱作言, 赖志春, 张文霞. 乳腺癌转移抑制因子BRMS1研究进展. 遗传, 2006, 28(9): 1141-1148.
[3] Mahamodhossen YA, Liu W, Zhou RR. Triple-negative breast cancer: new perspectives for novel therapies. Med Oncol , 2013, 30(6): 653-659.
[4] Alderton GK. Diagnosis: fishing for exosomes. Nat Rev Cancer , 2015, 15(8): 453-454.
[5] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin , 2011, 61(2): 69-90.
[6] Ludwig AK, Giebel B. Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol , 2012, 44(1): 11-15.
[7] Record M, Subra C, Silvente-Poirot S, Poirot M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochem Pharmacol , 2011, 81(10): 1171-1182.
[8] Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ. B lymphocytes secrete antigen-presenting vesicles. J Exp Med , 1996, 183(3): 1161-1172.
[9] Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem , 1987, 262(19): 9412-9420.
[10] Conde-Vancells J, Rodriguez-Suarez E, Embade N, Gil D, Matthiesen R, Valle M, Elortza F, Lu SC, Mato JM, Falcon-Perez JM. Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. J Proteome Res , 2008, 7(12): 5157-5166.
[11] Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol , 2007, 9(6): 654-659.
[12] Boelens MC, Wu TJ, Nabet BY, Xu BH, Qiu Y, Yoon T, Azzam DJ, Twyman-Saint Victor C, Wiemann BZ, Ishwaran H, Ter Brugge PJ, Jonkers J, Slingerland J, Minn AJ. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways. Cell , 2014, 159(3): 499-513
[13] Zhou WY, Fong MY, Min YF, Somlo G, Liu L, Palomares MR, Yu Y, Chow A, O’Connor STF, Chin AR, Yen Y, Wang YF, Marcusson EG, Chu PG, Wu J, Wu XW, Li AX, Li Z, Gao HL, Ren XB, Boldin MP, Lin PC, Wang SE. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell , 2014, 25(4): 501-515.
[14] Rak J. Organ-seeking vesicles. Nature , 2015, 527(1038): 312-313.
[15] Bysell H, Månsson R, Hansson P, Malmsten M. Microgels and microcapsules in peptide and protein drug delivery. Adv Drug Deliv Rev , 2011, 63(13): 1172-1185.
[16] Wu XB, Dong XY, Wu ZJ, Qu JG, Hou YD. A novel method for purification of recombinant adeno-associated virus vectors on a large scale. Chin Sci Bull , 2000, 45(19): 2071-2075. 吴小兵, 董小岩, 伍志坚, 屈建国, 侯云德. 一种快速高效分离和纯化重组腺病毒伴随病毒载体的方法. 科学通报, 2000, 45(19): 2071-2075.
[17] Milane L, Singh A, Mattheolabakis G, Suresh M, Amiji MM. Exosome mediated communication within the tumor microenvironment. J Control Release , 2015, 219: 278-294.
[18] Logozzi M, De Milito A, Lugini L, Borghi M, Calabrò L, Spada M, Perdicchio M, Marino ML, Federici C, Iessi E, Brambilla D, Venturi G, Lozupone F, Santinami M, Huber V, Maio M, Rivoltini L, Fais S. High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients. PLoS One , 2009, 4(4): e5219.
[19] Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol , 2008, 110(1): 13-21.
[20] Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban

Metrics

Comments

www.chinagene.cn
备案号：京ICP备09063187号

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 11

Recommended Articles

Metrics

Comments

[1]	Chunhui Ma, Haixu Hu, Lijuan Zhang, Yi Liu, Tianyi Liu. Establishment and verification of a digital PCR assay for the detection of CK19 expression in quantitative analysis of circulating tumor cell [J]. Hereditas(Beijing), 2023, 45(3): 250-260.
[2]	Dong Chang, Xiangxiang Liu, Rui Liu, Jianwei Sun. The role and regulatory mechanism of FSCN1 in breast tumorigenesis and progression [J]. Hereditas(Beijing), 2023, 45(2): 115-127.
[3]	Qiang Zhang, Mingliang Gu. Single-cell sequencing and its application in breast cancer [J]. Hereditas(Beijing), 2020, 42(3): 250-268.
[4]	Xinyuan Wang, Yu Zhang, Nan Yang, He Cheng, Yujie Sun. DNMT3a mediates paclitaxel-induced abnormal expression of LINE-1 by increasing the intragenic methylation [J]. Hereditas(Beijing), 2020, 42(1): 100-111.
[5]	Qichao Yu,Bin Song,Xuanxuan Zou,Ling Wang,Dequan Liu,Bo Li,Kun Ma. Analysis of normal tissues adjacent to the tumour-specific expressed genes in breast cancer [J]. Hereditas(Beijing), 2019, 41(7): 625-633.
[6]	Tonglu YU,Dongliang Cai,Genfeng Zhu,Xiaojuan Ye,Taishan Min,Hongyan Chen,Daru Lu,Haoming Chen. Effects of CSN4 knockdown on proliferation and apoptosis of breast cancer MDA-MB-231 cells [J]. Hereditas(Beijing), 2019, 41(4): 318-326.
[7]	Chuanbo Yao, Xin Zhou, Ceshi Chen, Qunying Lei, . The regulatory mechanisms and functional roles of the Hippo signaling pathway in breast cancer [J]. Hereditas(Beijing), 2017, 39(7): 617-629.
[8]	Zhenhua Hao, Wei Li. Muted protein is involved in the targeting of CD63 to large dense-core vesicles of chromaffin cells [J]. Hereditas(Beijing), 2016, 38(8): 718-723.
[9]	WU Xin-Gang PENG Shu-Bin HUANG Qian. Transcriptional regulation of breast cancer resistance protein [J]. HEREDITAS, 2012, 34(12): 1529-1536.
[10]	. Molecular mechanisms of regulation of estrogen receptor a expression level in breast cancer [J]. HEREDITAS, 2010, 32(3): 191-197.
[11]	WANG Jing, LI Yan-Hui, GUO Zheng, ZHU Jing, MA Wen-Cai, PENG Chun-Fang, LIU Qing. Finding finer functions for proteins related to breast cancer based on protein interaction network [J]. HEREDITAS, 2007, 29(9): 1061-1066.