Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.
Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.
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DOI：
10.1016/j.ymeth.2015.03.028
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发表时间：
2015-10-01
期刊：
影响因子：
--
通讯作者：
Tannetta DS
中科院分区：
文献类型：
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作者：
Dragovic RA;Collett GP;Hole P;Ferguson DJ;Redman CW;Sargent IL;Tannetta DS
胎盘灌注可产生大量的合胞体滋养层细胞外囊泡（STBEV）。
差速离心可分离出富含合胞体滋养层微囊泡（STBMV）和合胞体滋养层外泌体（STBEX）的制剂。
荧光纳米颗粒追踪分析（fl - NTA）可使用一种细胞外囊泡“标准品”进行优化。
fl - NTA利用特异性标志物胎盘碱性磷酸酶（PLAP）能够可靠地检测STBMV。
人类胎盘会向母体循环中释放多种类型和大小的合胞体滋养层（STB）细胞外囊泡（EV），这些囊泡具有多种生物活性。胎盘灌注技术能够分离这些STBEV，但传统的流式细胞术只能用于对大小约300nm及以上的细胞外囊泡进行表型分析。荧光纳米颗粒追踪分析（fl - NTA）有可能对大小约50nm及以上的细胞外囊泡进行表型分析，从而改进现有的表征技术。本研究的目的是从人胎盘灌注液（n = 8）中制备富含微囊泡和外泌体的组分，并改进fl - NTA对STBEV的检测。差速离心和过滤可有效去除新鲜胎盘灌注液中的污染红细胞，并使合胞体滋养层微囊泡（STBMV）组分（10000×g离心沉淀 - 10KP；纳米颗粒追踪分析测得的众数粒径为395 ± 12nm）沉淀，该组分富含合胞体滋养层标志物胎盘碱性磷酸酶（PLAP），以及使合胞体滋养层外泌体（STBEX）组分（150000×g离心沉淀 - 150KP；纳米颗粒追踪分析测得的众数粒径为147 ± 6nm）沉淀，该组分富含PLAP以及外泌体标志物Alix和CD63。通过免疫磁珠去除法确定的“标准”10KP和150KP混合样本（每个混合样本含四个样品）的PLAP阳性情况被用于优化fl - NTA的相机设置。单个的10KP和150KP样品（n = 8）分别有54.5 ± 5.7%（范围17.8 - 66.9%）和30.6 ± 5.6%（范围3.3 - 51.7%）为PLAP阳性。我们已经开发出一种从胎盘灌注液中富集STBMV和STBEX的可靠方法。我们还对fl - NTA的设置进行了标准化，并改进了对STBMV中PLAP阳性细胞外囊泡的测量。然而，fl - NTA在检测STBEX方面不如抗 - PLAP磁珠捕获法灵敏，这可能是由于STBEX的PLAP表面表达较低。这些重要的进展将有助于对STBMV和STBEX在正常和病理妊娠中的作用进行更详细的研究。
Placental perfusion yields large amounts of STBEV. Differential centrifugation isolates enriched preparations of STBMV and STBEX. fl-NTA can be optimised using an EV ‘standard’. fl-NTA reliably detects STBMV using the specific marker PLAP. The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n = 8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet – 10KP; NTA modal size 395 ± 12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet – 150KP; NTA modal size 147 ± 6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of ‘standard’ 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n = 8) were 54.5 ± 5.7% (range 17.8–66.9%) and 30.6 ± 5.6% (range 3.3–51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.