Platelet adhesion to endothelial cells in vitro, observation with video-enhanced contrast (VEC) microscopy

体外血小板与内皮细胞的粘附，用视频增强对比 (VEC) 显微镜观察

• 批准号: 10670601
• 负责人: TANAHASHI Norio
• 金额: $ 1.86万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670601/
• 关键词: endlthelial cell; platelet adhesion; thrombin; GPIIb; IIIa; endo thelial cell; platelet; adhesion

We examined whether or not platelets adhere to endothelial cells treated by thrombin in vitro using video enhanced contrast-differential interference contrast (VEC-DIC) microscopy, and also the role of platelet glycoprotein (GP) Iib/IIIa on platelet adhesion to endothelial cells.METHODS: VEC microscopy: The VEC equipment consisted of an inverted Nomarski microscope and a CCD camera coupled with an image processing processor, monitored at video image magnifications of up to x12000, and recorded on video tape. Experiment 1(N=10): Human aortic endothelial cells (HAEC) were cultured on the coverglass and put in the observation chamber of VEC-DIC microscopy. Human α thrombin 1U/ml with Hank's solution was superfused on HAEC for 30 minutes and washed out. Then, platelet rich plasma (PRP) separated from venous blood of healthy volunteers were superfused with an infusion pump at a low shear rate (10/sec) for 30 min and washed out. Interaction between platelets and endothelial cells was observe … More d by VEC-DIC microscopy and number of platelets adhered to endothelial cells were calculated. Experiment 2 (N=7): Following thrombin pretreatment on HAEC, PRP with TAK029 (1μM, GP Iib/IIIa antagonist) were superfused for 30 min and adhesion of platelets to endothelial cells were observed. Experiment 3 (N=7): As a control, PRP only were superfused for 30 min and adhesion of platelets to endothelial cells were observed. To quantify the degree of platelet adhesion to endothelial cells, number of adhering platelets in a field of 30 micrometer in length and breadth was counted. Consecutive 100 fields was counted and the average number was calculated.RESULTS: Experiment 1: Platelets adhered to endothelial cells in all experiments and microaggregates of platelets were seen. Average number of platelets adhering and aggragating to endothelial cells were 16.1±9.8/900μmィイD12ィエD1. Experiment 2: Platelets adhesion to endothelial cells were clearly suppressed. Average number of platelets adhering and aggregating to endothelial cells were 0.4±0.3/900μmィイD12ィエD1 (P<0.01, compared to Experiment 1). Experiment 3: Platelets adhesion to endothelial cells were scarecely seen, Average number of platelets adhering and aggregating to endothelial cells were 0.3±0.7/900μmィイD12ィエD1.CONCLUSION: The above results indicate that platelet adhered to thrombin-treated endothelial cells and GP Iib/IIIa plays an important role for platelets adhesion to endothelial cells in vitro. Less

我们在体外利用视频增强对比-差示干涉对比(VEC-DIC)显微镜检测了血小板是否与凝血酶处理的内皮细胞黏附，以及血小板糖蛋白(GP)IIb/IIIa在血小板与内皮细胞黏附中的作用。方法：VEC显微镜：VEC设备由一个倒置的Nomarski显微镜和一个与图像处理处理器耦合的CCD摄像机组成，以高达x12000的视频图像放大倍数监测，并记录在录像带上。实验一(N=10)：人主动脉内皮细胞(HAEC)在盖玻片上培养，置于VEC-DIC显微镜观察室中。人α凝血酶1U/ml与HANK‘s液在HAEC上灌流30min，洗净。然后，从健康志愿者静脉血中分离出富含血小板血浆(PRP)，用输液泵以低剪切率(10次/秒)灌流30min，洗净。…观察血小板与血管内皮细胞相互作用计算VEC-DIC显微镜下观察的天数和与血管内皮细胞黏附的血小板数。实验2(N=7)：凝血酶预内皮细胞后，用TAK0 2 9(1μM，GP IIb/IIa拮抗剂)灌流PRP 30min，观察血小板与内皮细胞的黏附。实验3(N=7)：对照组只灌流PRP 30min，观察血小板与内皮细胞的黏附情况。为了量化血小板与内皮细胞的黏附程度，在长度和宽度为30微米的视野中计算了黏附的血小板的数量。结果：实验1：所有实验均可见血小板与血管内皮细胞黏附，可见血小板微聚体。与内皮细胞黏附和加重的平均血小板数为16.1±9.8个/90 0μmィイD12ィエD1。实验2：血小板与血管内皮细胞的黏附受到明显抑制。与实验1相比，内皮细胞黏附和聚集的平均血小板数为0.4±0.3/90 0μmィイD12ィエd1(P<0.0 1)。实验3：血小板与内皮细胞黏附稀少，平均黏附数为0.3±0.7/900μmィイD12ィエD1。结论：凝血酶处理后的血管内皮细胞与血小板黏附，GP IIb/IIIa在血小板黏附中起重要作用。较少

项目成果

Itoh Y,Tomita N,Tanahashi N et al: "Platelet adhesion to Aortic endothelial cells in vitro after thrombin treatment: observation with video-enhanced contrast microscopy" Thrombosis Research. 91. 15-21 (1998)

Itoh Y，Tomita N，Tanahashi N 等人：“凝血酶治疗后体外血小板粘附到主动脉内皮细胞：用视频增强对比显微镜观察”血栓形成研究。

Itoh Y, Tanahashi N. et al: "Platelet adhesion to aortic endothelial cells in vitro after thrombin treatment: observation with video-enhance of contrast microscopy"Thromb. Res.. 91. 15-21 (1998)

Itoh Y、Tanahashi N. 等人：“凝血酶处理后体外血小板与主动脉内皮细胞的粘附：用视频增强对比显微镜观察”血栓。

Tanahashi N, et al: "Platelet adhesion to human brain microvascular-endothelial cells in vitro. Observation with video-enhanced contrast microscopy"Neurosci. Lett.. 274. 199-202 (1999)

Tanahashi N 等人：“体外血小板粘附到人脑微血管内皮细胞。用视频增强对比显微镜观察”Neurosci。

Tanahashi.N,Fukuuchi Y,et al: "Activated platelets adhere to endothelial cells in vitro-observation by VEC-DIC microscopy"Microcirculation annual. 15. 17-18 (1999)

Tanahashi.N，Fukuuchi Y，等人：“活化的血小板粘附于内皮细胞体外 - 通过 VEC-DIC 显微镜观察”微循环年度。