Proteomic analysis of exosomes isolated from human malignant pleural effusions
Proteomic analysis of exosomes isolated from human malignant pleural effusions
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DOI：
10.1165/rcmb.2003-0238oc
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发表时间：
2004-07-01
影响因子：
6.4
通讯作者：
Lambrecht, BN
中科院分区：
文献类型：
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作者：
Bard, MP;Hegmans, JP;Lambrecht, BN
外泌体是由各种细胞如造血细胞、上皮细胞和肿瘤细胞分泌的来自内体来源的膜囊泡。由肿瘤细胞分泌的外来体含有可能用于免疫目的的特异性抗原。我们的目的是确定外来体是否存在于人类癌性胸腔积液中，并确定其蛋白质组含量。通过蔗糖梯度离心纯化外泌体，并使用电子显微镜检查外泌体的浓度和纯度。蛋白质经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，基质辅助激光解吸电离飞行时间质谱和蛋白质印迹鉴定。外泌体存在于从患有间皮瘤（n = 4）、肺癌（n = 2）、乳腺癌（n = 2）和卵巢癌（n = 1）的患者获得的胸膜液中。如先前其他人所报道的，存在抗原呈递分子、细胞骨架蛋白和信号转导相关蛋白。鉴定了以前未报道的蛋白质（SNX 25、BTG 1、PEDF、血小板反应蛋白2）。不同类型的免疫球蛋白和补体因子大量存在于含有外泌体的蔗糖级分中。未观察到这些免疫球蛋白的外来体定向特异性。总之，蔗糖梯度超离心法可以从恶性胸腔积液中分离外来体。然而，胸膜液蛋白质，特别是免疫球蛋白是共分离的，可能会妨碍使用从恶性积液分离的外来体用于免疫治疗方案。
Exosomes are membrane vesicles from endosomal origin secreted by various cells such as hematopoietic, epithelial, and tumor cells. Exosomes secreted by tumor cells contain specific antigens potentially useful for immunotherapeutic purposes. Our aim was to determine if exosomes are present in human cancerous pleural effusions and to identify their proteomic content. Exosomes were purified by sucrose gradient ultracentrifugation, and electron microscopy was used to check both concentration and purity of exosomes. Proteins were separated by one-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and protein bands were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blotting. Exosomes were present in pleural fluid obtained from patients suffering from mesothelioma (n = 4), lung cancer (n = 2), breast cancer (n = 2), and ovarian cancer (n = 1). As previously reported by others, antigen-presenting molecules, cytoskeletal proteins, and signal transduction-involved proteins were present. Proteins not previously reported were identified (SNX25, BTG1, PEDF, thrombospondin 2). Different types of immunoglobulins and complement factors were abundantly present in the sucrose fractions containing exosomes. Exosome-directed specificity of these immunoglobulins was not observed. In conclusion, sucrose gradient ultracentrifugation allows isolation of exosomes from malignant pleural effusions. However, pleural fluid proteins and especially immunoglobulins are coisolated and may hamper the use of exosomes isolated from malignant effusion for immunotherapy programs.