适配子纤维蛋白靶向性验证及其抗凝活性测定

Fibrin-targeting Validation of Aptamer and its Anticoagulant Activity Determination

目的:验证适配子G81的纤维蛋白靶向性,评估适配子对凝血系统的影响。方法:以复钙法制备鼠源、人源体外纤维蛋白,将不同浓度Cy5.5标记的适配子溶液与之孵育,置于激光共聚焦显微镜下以固定的参数成像,用ImageJ软件进行相对荧光强度分析;将适配子G81溶液加入血浆中,通过倍比稀释法得到含浓度梯度适配子的血浆,采用SYSMEX CS-5100全自动血凝仪检测PT、APTT、TT,评估适配子G81对凝血功能的影响。结果:激光共聚焦显微镜显示适配子能与纤维蛋白结合,随着加入适配子量的增加其相对荧光强度逐渐增强,表明适配子可与纤维蛋白结合,统计分析提示荧光强度与适配子存在量效关系;人源、鼠源纤维蛋白结合的荧光强度无统计学差异(P>0.05)。在抗凝活性检测中,血浆中适配子G81浓度达到200 pmoL/mL时,各浓度统计分析结果均显示P>0.05,表明适配子对PT、APTT、TT的测量均没有统计学差异上的影响。结论:适配子G81具有纤维蛋白靶向性,且当加入的适配子剂量低于200 pmol/mL时对内、外源性凝血功能、凝血酶时间均无明显影响。

Objective: To verify the fibrin-targeting of aptamer G81 and evaluate its effects on the coagulation system. Methods:Fibrin was prepared in vitro and the aptamer labeled with Cy5.5 at different concentrations was incubated with it. Fluorescence intensity was observed under the laser confocal microscope and analyzed by ImageJ software. Then, the plasma containing the gradient concentration aptamer was obtained by the multiple dilution method and the aptamer G81 was added into the plasma. The prothrombin time(PT),activated partial thromboplastin time(APTT) and thrombin time(TT) were detected by the automatic coagulometer to evaluate the effects on the coagulation function. Results: Confocal laser microscopy showed that the aptamer could bind to fibrin, and the fluorescence intensity increased with the rise of the aptamer. Statistical analysis indicated that there was a dose-effect relationship between relative fluorescence intensity and aptamers. And there was no significance in the fluorescence intensity between human and mouse fibrin(P>0.05).In the detection of anticoagulant activity, there was no statistically significant difference between the concentration of aptamer G81 in plasma below 200 pmol/m L(P>0.05). Conclusion: The aptamer G81 is fibrin-targeting and has no obvious effects on endogenous and exogenous coagulation functions and thrombin time when below 200 pmol/mL of aptamer.