体外组织因子微颗粒的表达及其促凝活性的分析

doi:10.3969/j.issn.1673-8640.2016.04.006

检验医学 ›› 2016, Vol. 31 ›› Issue (4): 270-274.DOI: 10.3969/j.issn.1673-8640.2016.04.006

体外组织因子微颗粒的表达及其促凝活性的分析

邵妍妍¹, 丁秋兰², 戴菁², 陆晔玲², 许冠群², 张利伟², 沈韻², 王鸿利¹, 王学锋²

1. 上海交通大学医学院附属瑞金医院上海血液学研究所医学基因组国家重点实验室,上海200025
2.上海交通大学医学院附属瑞金医院检验科,上海 200025

收稿日期:2015-10-09 出版日期:2016-04-30 发布日期:2016-05-12
作者简介:null
作者简介：邵妍妍,女,1991年生,硕士,主要从事出血病和血栓病的基础和临床研究。

通讯作者：丁秋兰,联系电话：021-64370045。
基金资助:
Novo Nordisk Haemophilia Research Fund in China

Expression and procoagulant property of tissue factor-positive microparticles generated in vitro

SHAO Yanyan¹, DING Qiulan², DAI Jing², LU Yeling², XU Guanqun², ZHANG Liwei², SHEN Yun², WANG Hongli¹, WANG Xuefeng²

1. State Key Laboratory of Medical Genomics,Shanghai Institute of Hematology,Ruijin Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200025,China
2. Department of Clinical Laboratory,Ruijin Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200025,China

Received:2015-10-09 Online:2016-04-30 Published:2016-05-12

摘要/Abstract

摘要：

目的体外细胞系培养刺激获得组织因子微颗粒（TF⁺MP）并对其促凝活性进行研究。方法用不同浓度脂多糖（LPS）刺激人急性单核细胞白血病细胞系（THP-1）,梯度离心后重悬分离获得微颗粒（MP）;在正常人去MP血浆中加入MP,采用凝血酶生成试验分别检测组织因子（TF）和磷酯酰丝氨酸（PS）的凝血酶生成能力;采用ZYMUPHEN MP-TF试剂盒检测TF阳性的MP中TF的促凝活性,选用0.45和0.65 μm的滤膜滤过MP,分别检测直径< 0.45和< 0.65 μm的MP颗粒的促凝活性。结果凝血酶生成试验和发色底物功能检测结果显示体外表达的MP具有促凝活性。凝血酶生成试验结果表明MP的凝血活性与LPS的刺激呈浓度依赖性增强。直径> 0.65 μm的MP颗粒具有较强的促凝活性。结论体外经THP-1单核细胞系培养表达MP具有一定的促凝活性。

关键词: 组织因子微颗粒, 组织因子, 磷脂酰丝氨酸, 凝血酶生成试验

Abstract:

Objective To study the procoagulant property of tissue factor-positive microparticles（TF⁺MP） generated in vitro through the culture and stimulation of cell line. Methods Human acute monocytic leukemia cell line（THP-1） was stimulated by different concentrations of lipopolysaccharide（LPS）. Microparticle（MP）was separated by gradient centrifugation. Thrombin generation abilities of tissue factor（TF） and phosphatidylserine（PS） were determined by thrombin generation test by adding MP into MP-depleted plasma. ZYMUPHEN MP-TF kit was performed to determine TF procoagulant property. The 0.45 and 0.65 μm filters were selected to determine MP procoagulant properties（< 0.45 and < 0.65 μm）. Results Both thrombin generation test and chromogenic substrate test demonstrated a procoagulant property of MP generated in vitro. Thrombin generation test further showed that the procoagulant property of MP increased by the addition of LPS in a concentration-dependent manner. MP > 0.65 μm had a strong procoagulant property. Conclusions MP generated in vitro through the culture and stimulation of THP-1 demonstrates certain procoagulant property.

Key words: Tissue factor-positive microparticles, Tissue factor, Phosphatidylserine, Thrombin generation test

中图分类号:

R446.11

邵妍妍, 丁秋兰, 戴菁, 陆晔玲, 许冠群, 张利伟, 沈韻, 王鸿利, 王学锋. 体外组织因子微颗粒的表达及其促凝活性的分析[J]. 检验医学, 2016, 31(4): 270-274.

SHAO Yanyan, DING Qiulan, DAI Jing, LU Yeling, XU Guanqun, ZHANG Liwei, SHEN Yun, WANG Hongli, WANG Xuefeng. Expression and procoagulant property of tissue factor-positive microparticles generated in vitro[J]. Laboratory Medicine, 2016, 31(4): 270-274.

图/表 4

图1 PRP/MP激活剂下不同LPS刺激浓度分离得到的MP加入去MP正常人混合血浆时的凝血酶生成量

图2 PRP/MP激活剂下不同LPS刺激浓度分离得到的MP加入去MP正常人混合血浆时的凝血酶生成各项指标

表1 ZYMUPHEN MP-TF活性检测

标本	TF活性（pg/mL）	细胞数校正后TF活性（pg/10⁶细胞）
MP-0.1 μg/mL LPS	29.46	23.38
MP-1 μg/mL LPS	27.61	24.54
MP-10 μg/mL LPS	20.21	19.97
MP-No LPS	3.47	2.27

图3 经0.45和0.65 μm孔径的滤膜滤过后MP的吸光度变化

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体外组织因子微颗粒的表达及其促凝活性的分析

Expression and procoagulant property of tissue factor-positive microparticles generated in vitro

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