天津医药

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大鼠循环血中血小板和内皮细胞来源微囊泡的检测

张梦晓1,2,尚曼2,张琦2,王瑶2,吴艳娜2,宋君秋2,刘艳霞3   

  1. 1. 蚌埠医学院
    2. 天津医科大学
    3. 天津医科大学基础医学院
  • 收稿日期:2013-10-28 修回日期:2013-12-27 出版日期:2014-04-15 发布日期:2014-04-15
  • 通讯作者: 刘艳霞

Detection of Platelets and Endothelial Cell-Derived Microvesicles in Rat Peripheral Blood

  • Received:2013-10-28 Revised:2013-12-27 Published:2014-04-15 Online:2014-04-15

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摘要： 【摘要】 目的 建立流式细胞术对大鼠循环血中细胞微囊泡(MVs)的检测方法，对心肌缺血预适应(IPC)处理大鼠循环血中血小板来源的MVs(PMVs)及内皮细胞来源的MVs(EMVs)进行测定。方法 大鼠经腹主动脉取血，枸橼酸钠抗凝，室温下经两步离心得无血小板血浆(PFP)。PFP中分别加入FITC标记的血小板表面标志物CD61单抗或PE标记的内皮细胞表面标志物CD144单抗，采用1 μm标准微球做粒径对照，2 μm标准微球用于计数，流式细胞仪进行检测。结果 3.5 %枸橼酸钠与血液按体积比1:4混匀，离心后可得均匀、清亮的上清。PFP中1 μm以下粒子信号占全部信号的99 %以上。PMVs和EMVs分别为CD61阳性和CD144阳性的粒子，直径均小于1 μm。IPC处理大鼠循环血中PMVs浓度为4053±1987个/μL，EMVs浓度为4870±825个/μL。结论 成功建立和优化了流式细胞术测定大鼠循环血中MVs的方法，并通过对IPC处理大鼠循环血中PMVs和EMVs的测定对MVs的检测方法进行了验证。

关键词: 血小板, 内皮细胞, 流式细胞术, 大鼠, Wistar, 细胞微囊泡, 缺血预适应

Abstract: [Abstract] Objective To establish a flow-cytometric method for detection of microvesicles (MVs) in rat peripheral blood, detect platelet-derived MVs (PMVs) and endothelial cell-derived MVs (EMVs) in blood from ischemic preconditioning (IPC) treated rats. Methods Blood was withdrawn from rat abdominal aorta and anticoagulated with sodium citrate. Platelet-free plasma (PFP) was obtained after two centrifugations at room temperature. PFP was incubated with FITC-conjugated mouse anti-rat CD61 or PE-conjugated mouse anti-rat CD144. Standard beads in diameter 1 and 2 μm were used for size calibration and absolute counting, respectively. Analysis was performed on flow cytometer. Results When 3.5% sodium citrate was blent with blood according to the volume ratio of 1:4, clear supernatant was obtained after centrifugation. Signals of particles smaller than 1 μm accounted for more than 99% of all. PMVs and EMVs were CD61 positive and CD144 positive, respectively. Their diameters were smaller than 1 μm. The concentration of PMVs and EMVs in peripheral blood from IPC treated rats was 4053±1987/μL and 4870±825/μL, respectively. Conclusion The method for MVs detection by flow cytometry was successfully established and optimized, and verified through detecting PMVs and EMVs in peripheral blood from IPC treated rats.

Key words: blood platelets, endothelial cells, flow cytometry, rats, Wistar, microvesicels, ischemic preconditioning