JoVE Logo
发表
联系我们

登录

需要订阅 JoVE 才能查看此. 登录或开始免费试用。

本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

该协议细节的方法分离细胞外囊泡（电动汽车），从细胞中释放的小的膜的颗粒，从少至10微升血清样品。这种方法绕过需要超速离心，需要测定时间仅几分钟，并且使电动车从有限体积的样品的分离。

摘要

胞外囊泡（电动汽车），从不同类型的细胞释放的膜的颗粒，持有用于临床应用的巨大潜力。它们含有核酸和蛋白质的货物和正日益被视为间通讯双方真核生物和原核生物细胞利用的手段。然而，由于它们的尺寸小，对电动汽车的隔离电流协议往往费时，繁琐，并且需要较大的样品体积和昂贵的设备，如超速离心。为了解决这些限制，我们开发了一种基于纸张免疫平台，用于分离电动汽车的亚组是容易的，高效的，并且需要的样品体积低至10微升。生物样品可以直接吸移到已经被化学修饰以捕获分子具有高亲和力的特定的EV表面标记纸测试区。我们验证该测定通过使用扫描电子显微镜（SEM），纸基酶联immunosorben吨测定（P-ELISA），和转录组分析。这些文件为基础的设备将使临床和研究设置电动车的研究，以帮助促进我们的健康和疾病EV功能的理解。

引言

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.

EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met.

We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

Access restricted. Please log in or start a trial to view this content.

研究方案

操作步骤的总体框图如图1中提供。使用道德实践，我们收集了来自健康受试者的血液样本，并通过台中荣民总医院（TCVGH），台中，台湾在IRB批准协议获得房水样本的患者（ IRB TCVGH号CF11213-1）。

1.制造纸张的设备

  1. 切色谱纸放入5mm的圆的直径，以提供相同的布局为一个96孔微量滴定板。夹着这些纸片具有两个聚苯乙烯片材与注册的通孔，和层压体。
  2. 修改使用在下面的步骤28中所述的化学缀合方法纸装置。
    1. 处理纸的设备用氧等离子体（100毫瓦，1％的氧气，30秒）在等离子体室中。
      注意:用3-巯丙基三甲氧基硅烷和 N-γ-马来酰亚胺琥珀酰亚胺工作时特别小心，必须采取酯（GMBS），因为它们都是湿气敏感和毒性。戴防护手套，避免吸入或与皮肤和眼睛接触。保持股市瓶子盖紧并允许它打开，以避免冷凝水之前平衡至RT。或者，打开股票瓶内充氮的手套袋中或框。分装成小药瓶，以避免频繁开股票瓶。
    2. 立即处理的孵育在4％（体积/体积）的3-巯基丙基三甲氧基硅烷的乙醇（200标准强度）为30分钟，溶液纸装置。
    3. 冲洗纸装置，用乙醇和孵化它们与0.01微摩尔/毫升GMBS在乙醇中15分钟。
    4. 冲洗，用乙醇（200标准强度），并培育纸装置用10μg/ ml的抗生物素蛋白在磷酸盐缓冲盐水（PBS），在4℃的溶液1小时。如果需要储存在4℃下，并在4周内使用。
  3. 湿纸的每个测试区与10微升的PBS含有1％（重量/体积）牛血清白蛋白（BSA）为察觉10微升生物素化的捕获分子3×10分钟前，3×10分钟。使用抗CD63抗体（20微克/毫升的PBS含有1％（重量/体积）BSA和0.09％（重量/体积）叠氮化钠）或膜联蛋白V（1:20，体积/体积在膜联蛋白V结合缓冲液），为捕获分子。
  4. 冲洗掉，用10微升相应的PBS含有1％（重量/体积）BSA或膜联蛋白V结合缓冲液进行3×1分钟，分别未结合的抗CD63抗体或膜联蛋白V的分子。

2.血清和房水样品采集与处理

  1. 血清集合。
    1. 在血清分离管中收集10毫升外周血通过静脉穿刺，轻轻颠倒试管5次。设置该管处于垂直位置，并等待30分钟。
    2. 离心机在1200×g下15分钟。再次传输从顶层血清到一个干净的试管中，离心以3000×g的30分钟。
    3. 通过0.8微米FIL通过上清器。保持样品在-80℃下直到使用。
  2. 房水的收集:收集房水样本的患者可直接通过侵入性操作和储存样品在-80°C，直到使用诊断眼科医生。

3.隔离外囊泡

  1. 点样到每个纸测试区以5微升/分钟的速率。
  2. 冲洗掉未结合的电动汽车用10微升相应的PBS含有1％（重量/体积）BSA或膜联蛋白V结合缓冲液进行3×1分钟，用于分别官能用抗CD63抗体或膜联蛋白V的分子，造纸设备。执行以下下游试验。

4.下游测定实施例1:扫描Electromicrographs

  1. 修复捕获使用10微升0.5×Karnovsky固定液10分钟官能试纸测试区的电动车。
  2. 冲洗了充足的PBS样品2×5分钟。
  3. 脱水样品随后35％乙醇10分钟，50％乙醇为2×10分钟，70％乙醇中2×10分钟，95％乙醇为2×10分钟，100％乙醇为4×10分钟。
  4. 临界干燥和溅射涂层与钯/金的样品中，并且检查使用在低电子加速电压操作的扫描型电子显微镜（〜5千伏）。

5.下游分析实施例2:基于纸张的ELISA

  1. 在PBS 1000稀释含电动车捕捉每一个测试区:添加含有抗CD9一抗5微升溶液中1。等待1分钟。
  2. 冲洗样品用30ml PBS中振摇在100rpm下30秒。
  3. 添加5微升含有溶液辣根过氧化物酶（HRP）为1 - 连接的二级抗体:1,000稀释在PBS中，并等待1分钟。
  4. 冲洗样品用30ml PBS中振摇在100rpm下30秒。
  5. 添加含有1 5微升比色底物:过氧化氢的1体积比ð3,3'，5,5'-四甲基联苯胺（TMB）和扫描使用台式扫描器。

6.下游试验例3:RNA分离

  1. 浸泡样品中35微升的聚乙烯吡咯烷酮为基础的RNA分离援助和265微升裂解/结合缓冲液裂解捕获电动车。涡大力。
  2. 加在分离试剂盒的裂解物提供30微升匀浆。涡大力和雪藏了10分钟。
  3. 使用在下面的步骤中描述的酸苯酚 - 氯仿分离提取的RNA。
    1. 取330微升酚 - 氯仿从瓶底层并添加到匀浆/溶胞产物。涡大力。
    2. 离心以10,000×g离心5分钟。水性（上部）相转移到一个干净的试管，并注意除去的体积。
  4. 沉淀的总RNA使用本身是在前面的步骤和共除去水相的1.25倍的体积的体积的100％乙醇llect在预热至95℃的洗提溶液的核糖核酸。
  5. 浓缩和进一步纯化根据生产商的方案使用RNA净化试剂盒的RNA。

Access restricted. Please log in or start a trial to view this content.

结果

造纸设备的隔离电动汽车的亚组的能力有效地依靠其敏感性和特异性识别的EV表面标志。纸纤维与捕获分子的稳定修饰是通过使用抗生物素蛋白-生物素化学如别处28-30所描述实现的。化学缀合的物理吸附方法的有效性，并且在使用基于荧光的读数评估。纸张测试区被以下置换为20微克/毫升的荧光团R-藻红蛋白缀合的生物素分子（PE-生物素），在步骤1.3协议步骤1），所不同的捕获分子制备。...

Access restricted. Please log in or start a trial to view this content.

讨论

为胞外囊泡的子组的成功分离的最重要的步骤是:1）一个良好的纸张选择; 2）稳定和高的纸纤维的表面上的捕获分子的覆盖范围; 3）样品的妥善处理; 4）一般实验室卫生习惯。

多孔材料已经被用于许多廉价且设备无测定。它们可以具有可调孔径，多用途功能，成本低和高的表面与体积之比，允许液体的被动的芯吸。我们选择色谱纤维素纸1级主要针对其适当的孔径和低蛋白吸?...

Access restricted. Please log in or start a trial to view this content.

披露声明

作者宣称，他们有没有竞争的财务权益。

致谢

这项工作是由台湾国科会grants- NSC 99-2320-B-007-005-MY2（CC）和NSC 101-2628-E-007-011-MY3（CMC），以及荣总部分支持医院和台湾联合研究计划大学系统（CC）。

Access restricted. Please log in or start a trial to view this content.

材料

NameCompanyCatalog NumberComments
Chromatography PaperGE Healthcare Life Sciences3001-861Whatman® Grade 1 cellulose paper
(3-Mercaptopropyl) trimethoxysilaneSigma Aldrich175617This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.
EthanolFisher ScientificBP2818Absolute, 200 Proof, molecular biology grade
Bovine serum albumin (BSA)BioShop Canada Inc.ALB001Often referred to as Cohn fraction V.
N-γ-maleimidobutyryloxy succinimide ester (GMBS)Pierce Biotechnology22309GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.
AvidinPierce Biotechnology31000NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin
Biotinylated mouse anti-human anti-CD63Ancell215-030clone AHN16.1/46-4-5
biotinylated annexin VBD Biosciences556418Annxin V has a high affinity for phosphatidylserine (PS)
Primary anti-CD9 and secondary antibodySystem BiosciencesEXOAB-CD9A-1The secondary antibody is horseradish peroxidise-conjugated
Serum separation tubesBD Biosciences367991Clot activator and gel for serum separation
Annexin V binding bufferBD Biosciences55645410x; dilute to 1x prior to use.
TMB substrate reagent setBD Biosciences555214The set contains hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB)
[header]
RNA isolation kitLife TechnologiesAM1560MirVana RNA isolation kit
Polyvinylpyrrolidone-based RNA isolation aidLife TechnologiesAM9690Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.
RNA cleanup kitQiagen Inc.74004MinElute RNA cleanup kit is designed for purification of up to 45 μg RNA.
Plasma chamberMarch InstrumentsPX-250
Scanning electron microscopeHitachi Ltd.S-4300
Desktop scannerHewlett-Packard CompanyPhotosmart B1108-bit color images were captured. Cameras and smart phones may be also used.
Image-record systemJ&H Technology CoGeneSys G:BOX Chemi-XX816-bit fluroscence images were captured. Fluroscence microscopes may be also used.

参考文献

  1. Caby, M. P., Lankar, D., Vincendeau-Scherrer, C., Raposo, G., Bonnerot, C. Exosomal-like vesicles are present in human blood plasma. Int. Immunol. 17, 879-887 (2005).
  2. Lasser, C., et al. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J. Transl. Med. 9, 9(2011).
  3. Raj, D. A. A., Fiume, I., Capasso, G., Pocsfalvi, G. A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes. Kidney Int. 81, 1263-1272 (2012).
  4. Wiggins, R. C., Glatfelter, A., Kshirsagar, B., Brukman, J. Procoagulant activity in normal human-urine associated with subcellular particles. Kidney Int. 29, 591-597 (1986).
  5. Admyre, C., et al. Exosomes with immune modulatory features are present in human breast milk. J. Immunol. 179, 1969-1978 (2007).
  6. Keller, S., Ridinger, J., Rupp, A. K., Janssen, J. W. G., Altevogt, P. Body fluid derived exosomes as a novel template for clinical diagnostics. J. Transl. Med. 9, 86(2011).
  7. Asea, A., et al. Heat shock protein-containing exosomes in mid-trimester amniotic fluids. J. Reprod. Immunol. 79, 12-17 (2008).
  8. Bard, M. P., et al. Proteomic analysis of exosomes isolated from human malignant pleural effusions. Am. J. Resp. Cell Mol. Biol. 31, 114-121 (2004).
  9. Perkumas, K. M., Hoffman, E. A., McKay, B. S., Allingham, R. R., Stamer, W. D. Myocilin-associated exosomes in human ocular samples. Exp. Eye Res. 84, 209-212 (2007).
  10. Anderson, H. C., Mulhall, D., Garimella, R. Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis. Lab. Invest. 90, 1549-1557 (2010).
  11. Montecalvo, A., et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood. 119, 756-766 (2012).
  12. Grange, C., et al. Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer Res. 71, 5346-5356 (2011).
  13. Jaiswal, R., et al. Microparticle-associated nucleic acids mediate trait dominance in cancer. Faseb. J. 26, 420-429 (2012).
  14. Thery, C., Clayton, A., Amigorena, S., Raposo, G. Current protocols in cell biology. Morgan, K. K. (UNIT 3.22), John Wiley. New York, NY. (2006).
  15. Rood, I. M., et al. Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int. 78, 810-816 (2010).
  16. Taylor, D. D., Gercel-Taylor, C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110, 13-21 (2008).
  17. Witwer, K. W., et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracellular Vesicles. 2, 20360(2013).
  18. Fernandez-Llama, P., et al. Tamm-Horsfall protein and urinary exosome isolation. Kidney Int. 77, 736-742 (2010).
  19. Alvarez, M. L., Khosroheidari, M., Ravi, R. K., DiStefano, J. K. Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int. 82, 1024-1032 (2012).
  20. Chen, C., et al. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab. Chip. 10, 505-511 (2010).
  21. Davies, R. T., et al. Microfluidic filtration system to isolate extracellular vesicles from blood. Lab. Chip. 12, 5202-5210 (2012).
  22. Chen, C., et al. Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles. Microfluid. Nanofluid. 16, 849-856 (2014).
  23. Lamparski, H. G., et al. Production and characterization of clinical grade exosomes derived from dendritic cells. J. Immunol. Methods. 270, 211-226 (2002).
  24. Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants. J. Immunol. Methods. 338, 21-30 (2008).
  25. Carrilho, E., Martinez, A. W., Whitesides, G. M. Understanding wax printing: a simple micropatterning process for paper-based microfluidics. Anal. Chem. 81, 7091-7095 (2009).
  26. Dungchai, W., Chailapakul, O., Henry, C. S. Electrochemical detection for paper-based microfluidics. Anal. Chem. 81, 5821-5826 (2009).
  27. Glavan, A. C., et al. Omniphobic 'R-F paper' produced by silanization of paper with fluoroalkyltrichlorosilanes. Adv. Funct. Mater. 24, 60-70 (2014).
  28. Usami, S., Chen, H. H., Zhao, Y. H., Chien, S., Skalak, R. Design and construction of a linear shear-stress flow chamber. Ann. Biomed. Eng. 21, 77-83 (1993).
  29. Murthy, S. K., Sin, A., Tompkins, R. G., Toner, M. Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers. Langmuir. 20, 11649-11655 (2004).
  30. Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A., Ligler, F. S. Comparison of chemical cleaning methods of glass in preparation for silanization. Biosens. Bioelectron. 14, 683-688 (1999).
  31. Thery, C., Zitvogel, L., Amigorena, S. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569-579 (2002).
  32. Scanu, A., et al. Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins. J. Leukocyte Biol. 83, 921-927 (2008).
  33. Inal, J. M., et al. Microvesicles in health and disease. Arch. Immunol. Ther. Ex. 60, 107-121 (2012).
  34. Fridley, G. E., Holstein, C. A., Oza, S. B., Yager, P. The evolution of nitrocellulose as a material for bioassays. Mrs Bull. 38, 326-330 (2013).
  35. Gyorgy, B., et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell. Mol. Life Sci. 68, 2667-2688 (2011).
  36. Missoum, K., Belgacem, M. N., Bras, J. Nanofibrillated cellulose surface modification: a review. Materials. 6, 1745-1766 (2013).
  37. Kalia, S., Boufi, S., Celli, A., Kango, S. Nanofibrillated cellulose: surface modification and potential applications. Colloid Polym. Sci. 292, 5-31 (2014).

Access restricted. Please log in or start a trial to view this content.

转载和许可

请求许可使用此 JoVE 文章的文本或图形

请求许可

探索更多文章

98 ELISA

This article has been published

Video Coming Soon

JoVE Logo

政策

使用条款

隐私

联系我们

向图书馆推荐

JoVE 新闻简报

科研

教育

发表

图书馆员

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有，本公司不涉及任何医疗业务和医疗服务。