人白细胞-HUVEC粘附的表征：细胞制备方法的影响。,Journal of Immunological Methods

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人白细胞-HUVEC粘附的表征：细胞制备方法的影响。
Journal of Immunological Methods ( IF 1.6 ) Pub Date : 2017-02-09 , DOI: 10.1016/j.jim.2017.01.013
Franca Marino ₁ , Laura Schembri ₁ , Emanuela Rasini ₁ , Monica Pinoli ₁ , Angela Scanzano ₁ , Alessandra Luini ₁ , Terenzio Congiu ₂ , Marco Cosentino ₁

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目的样品处理以获得分离的粒细胞是体外研究中的关键步骤，通常是必需步骤。我们通过流式细胞仪和显微镜技术（光学显微镜[OM]和扫描电子显微镜[SEM]）研究了粒细胞-内皮粘附和样品处理的作用。方法通过共培养方法，我们分析了源自两种不同血液样本（新鲜静脉血[FB]和血沉棕黄层[BC]）的人白细胞对人脐静脉内皮细胞（HUVEC）的粘附单层。通过流式细胞术分析培养的HUVEC的粘附分子表达，同时通过SEM评估形态变化。通过流式细胞术以及OM和SEM评估细胞粘附。结果HUVEC在静息条件下表达粘附分子ICAM-1，VCAM-1和E-选择素，并通过TNF-α（0.1-10ng / ml）和LPS（1μg/ ml）刺激上调其表达。SEM分析表明，两种刺激均能深刻影响细胞形态。流式细胞术评估细胞黏附性表明，两种制剂中细胞黏附于HUVEC单层的能力差异很大，在所有细胞亚群中FB的黏附性最低。最后，与在FB或BC中鉴定的细胞相比，分离的粒细胞能够更多地粘附于HUVEC单层，并且在用10ng / mlTNF-α激活HUVEC期间粘附力增加。

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Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods.

OBJECTIVE Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. METHODS By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM. RESULTS HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1-10ng/ml) as well as with LPS (1μg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10ng/ml of TNF-α. CONCLUSION Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.

更新日期：2019-11-01

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