摘要
目的研究生理性流动条件下血小板在玻璃表面的聚集行为。方法采用软光刻工艺加工聚二甲基硅氧烷(polydimethylsiloxane,PDMS)-玻璃微通道芯片。将抗凝人外周全血以300、1500 s^(-1)壁剪切率流过微通道芯片,倒置荧光显微镜拍摄荧光标记血小板在微通道玻璃表面形成的血小板聚集体荧光图像,通过图像分析得到血小板聚集体数量、平均尺寸、表面覆盖率和平均荧光强度。分别对玻璃表面进行亲水性处理、牛血清白蛋白(bovine serum albumin,BSA)封闭和胶原纤维蛋白修饰处理,调整血液样品红细胞比容(hematocrit,Hct),用抗血小板制剂处理血液样品,观察上述条件下血小板在玻璃表面的聚集行为;比较分析健康人群和糖尿病患者的血小板聚集情况。结果流动条件下,血小板在玻璃表面发生聚集形成三维血小板聚集体;血小板聚集依赖于壁剪切率大小、玻璃表面亲疏水性和Hct;血小板聚集主要由血小板膜糖蛋白GPIIb/IIIa-纤维蛋白原和ADP-P2Y_(12)受体通路调控;血小板在玻璃表面的聚集能够反映糖尿病患者血小板的高活化状态。结论在300、1500 s^(-1)流动条件下,血小板在玻璃表面的聚集与剪切率、蛋白吸附、血小板相关受体和血小板自身活化状态相关。本文建立了一种无需额外黏附蛋白修饰的微流控血小板功能分析的新模型,可用于临床血小板功能评估。
Objective To investigate platelet aggregation on glass surface under physiological flow condition.Methods The polydimethylsiloxane(PDMS)-glass microchannel chips were fabricated by soft lithography.Anticoagulant human peripheral whole blood was flowed through the microchannel chip at flow shear rate of 300 s^(-1) and 1500 s^(-1),respectively.The fluorescence images of platelet aggregates formed on glass surface at the bottom of the microchannel were captured after 150 s using an inverted fluorescence microscope.The number of platelet aggregates,average size,surface coverage and average fluorescence intensity were quantified by image analysis.The glass surface was treated with oxygen plasma,BSA blocking or collagen modification to establish different surfaces for platelet aggregation.The hematocrit(Hct)of blood sample was adjusted,and the whole blood was treated with different anti-platelet agents.The platelet aggregation on glass surface was observed under the above experimental conditions.The platelet aggregations in healthy people and diabetic patients were also analyzed.Results Under the flow condition,platelet aggregation on glass surface was three-dimensional.Platelet aggregation was dependent on wall shear rate,the hydrophilicity of glass surface and Hct,and was mainly regulated by GPIIb/IIIa-fibrinogen and ADP-P2Y_(12) receptor pathways.The aggregation of platelets on the glass surface could also reflect the high activity of platelets in diabetic patients.Conclusions At the flow conditions of 300 s^(-1) and 1500 s^(-1),platelet aggregation on glass surface is related to flow rate,protein adsorption,platelet related receptors and platelet activation state.In this study,a new model for microfluidic platelet function analysis without additional adhesion protein modification was established,and it could be used for clinical evaluation of platelet function.
作者
张天聪
陈丹
廖娟
邓素容
米永华
刘占术
何菲
胡煜琳
李远
ZHANG Tiancong;CHEN Dan;LIAO Juan;DENG Surong;MI Yonghua;LIU Zhanshu;HE Fei;HU Yulin;LI Yuan(Central Laboratory,Yongchuan Hospital of Chongqing Medical University,Chongqing 402160,China)
出处
《医用生物力学》
CAS
CSCD
北大核心
2022年第3期425-432,共8页
Journal of Medical Biomechanics
基金
国家自然科学基金项目(11702047)
重庆市社会事业与民生保障科技创新专项(cstc2017shmsA130009)
重庆市医学科研计划项目(2017MSXM079)。
