School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia;
School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia.
J Extracell Vesicles. 2016 Apr 15;5:25355. doi: 10.3402/jev.v5.25355. eCollection 2016.
Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.
细胞分泌的囊泡（EV）是一种膜性颗粒（直径 30-1000nm）。EV 可分为 2 个亚群，即外泌体（起源于内体膜的出芽）和微囊泡（起源于质膜的出芽），这 2 个亚群都具有重要的生物学功能。由于这两种颗粒都含有来源于其原始细胞的表面蛋白，因此它们在血液中的检测可能有助于疾病的诊断和预后。我们使用抗体微阵列（DotScan），根据与固定化抗体的结合模式，比较了活癌细胞及其 EV 的表面蛋白谱。最初，使用源自癌细胞系 LIM1215（结直肠癌）和 MEC1（B 细胞慢性淋巴细胞白血病；CLL）的 EV 进行了测定优化。分别使用针对 EpCAM（CD326）和 CD19 的生物素化抗体来检测捕获的颗粒，通过增强化学发光进行检测。随后，我们使用这种方法对来自 CLL 患者血浆的 CD19+EV 进行了分析。这些 EV 表达了来自同一患者 CLL 细胞的一部分（~40%）检测到的蛋白：CD5、CD19、CD31、CD44、CD55、CD62L、CD82、HLA-A、B、C、HLA-DR 表达中等或高水平；CD21、CD49c、CD63 表达低水平。在年龄和性别匹配的健康个体的血浆 EV 中未检测到这些蛋白。
