目的 建立血小板细胞膜色谱模型, 考察不同温度下抗血小板聚集药物在该色谱柱上的保留行为,模拟药物在正常和发热病理状态下与血小板的相互作用。方法 通过物理吸附法,构建血小板细胞膜色谱固定相,湿法装柱,制成血小板细胞膜色谱柱,BCA蛋白浓度测定试剂盒测定蛋白含量,Na+、K+-ATPase检测试剂盒测定生物活性,应用该色谱模型,考察色谱柱的特异性及在35.0~42.0 ℃温度范围下药物的保留特性。结果 构建的血小板细胞膜色谱模型其血小板ATP酶活性值为0.214, 固定前血小板膜蛋白质量浓度为0.340 9 mg·mL-1,固定后血小板膜蛋白质量浓度为0.080 5 mg·mL-1。3个抗血小板聚集药物氯吡格雷、双嘧达莫和西洛他唑在血小板细胞膜色谱柱和空白硅胶柱上保留特征有较大差异,36.0 ℃时3个药物在血小板细胞膜色谱柱上的保留时间均为最大值,然后随温度的升高,保留时间均呈下降的趋势。结论 成功构建了血小板细胞膜色谱模型,并首次研究了不同温度下抗血小板聚集药物在该色谱模型上的保留特性,模拟了正常和发热体温时抗血小板聚集药物的色谱保留行为。
Abstract
OBJECTIVE To establish a platelet cell membrane chromatographic model and investigate the retention behaviors of anti-platelet aggregation drugs on chromatographic column at different temperatures, and simulate the interactions between drugs and platelets under normal and febrile pathological conditions. METHODS The platelet cell membrane chromatographic stationary phase was constructed by physical adsorption method. The column was packed with wet method. The protein content was determined by BCA protein concentration assay kit. The biological activity was determined by Na+, K+-ATPase assay kit. The chromatographic model was used to investigate the specificity of the column and the retention characteristics of drugs in the temperature range of 35.0-42.0 ℃. RESULTS The activity of platelet ATPase was 0.214, and the concentration of platelet membrane protein was 0.340 9 mg·mL-1 before bonding and 0.080 5 mg·mL-1 after bonding. The retention characteristics of clopidogrel, dipyridamole and cilostazole on platelet cell membrane chromatographic column and blank silica gel column were quite different. The retention time of the three drugs on platelet cell membrane chromatographic column was the maximum at 36.0 ℃, and then decreased with the increase of temperature. CONCLUSION A platelet cell membrane chromatographic model is successfully constructed, and the retention characteristics of antiplatelet aggregates at different temperatures are studied for the first time. The chromatographic retention behaviors of antiplatelet aggregates at normal and febrile body temperatures are simulated.
关键词
细胞膜色谱 /
血小板细胞膜色谱固定相 /
高效液相色谱法 /
抗血小板药物
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Key words
cell membrane chromatography /
platelet cell membrane chromatographic stationary phase /
high performance liquid chromatography /
antiplatelet drug
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中图分类号：
R917
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脚注
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基金
湖北省自然科学基金项目资助(2018CFC868); 湖北省教育厅科研计划指导性项目资助(B2017113);湖北省十堰市科技局引导性项目资助(17Y05)
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