凝血酶智能响应型纳米探针用于动脉血栓性疾病诊断的实验研究  

Application of thrombin responsive nano-probe in diagnosis of arterial thrombotic disease

作　　者：高姗 许梦琪[1] 杨宁 刘静[2] 谢晓烨 方岩 田磊[2] 刘建伟[2] 王亚斌[1] 曹丰[4] Gao Shan;Xu Mengqi;Yang Ning;Liu Jing;Xie Xiaoye;Fang Yan;Tian Lei;Liu Jianwei;Wang Yabin;Cao Feng(Department of Cardiology,Chinese PLA General Hospital No.2 Medical Center,Beijing 100853,China)

机构地区：[1]解放军总医院第二医学中心心血管内科,北京100853 [2]解放军总医院老年医学研究所,北京100853 [3]解放军总医院第一医学中心心血管内科,北京100853 [4]解放军总医院第二医学中心,北京100853

出　　处：《中华老年心脑血管病杂志》2021年第5期527-530,共4页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：国家自然科学基金(91939303);2019解放军总医院军事医学创新项目(CX19028)。

摘　　要：目的应用凝血酶智能响应型纳米探针,实现颈动脉血栓的体内可视化预测,为血栓形成的早期诊断提供新策略。方法将超顺磁性氧化铁与凝血酶响应肽及Cy5.5-N-羟基琥珀酰亚胺酯(Cy5.5 NHS)共价偶联构建探针。检测不同浓度探针(0μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml、200μg/ml)对人脐静脉内皮细胞的毒性。近红外荧光成像分析加入不同浓度凝血酶的探针荧光强度(实验组:凝血酶浓度分别为0.5 U/ml、1 U/ml、10 U/ml、50 U/ml;空白对照组为0 U/ml;阴性对照组为50 U/ml,并加入100μg比伐芦定)。制备小鼠颈动脉血栓模型,并对血栓组和对照组小鼠颈动脉组织切片进行免疫荧光染色,以探究探针的凝血酶靶向性。结果不同探针浓度的细胞存活率比较无统计学差异(P>0.05)。不同浓度实验组探针荧光强度分别为空白对照组的17.64、34.24、55.01和64.65倍(P<0.01),阴性对照组与空白对照组探针荧光强度比较无显著差异(P>0.05)。血栓组小鼠病变局部凝血酶水平较对照组显著升高。结论本研究成功构建凝血酶智能响应型纳米探针,实现颈动脉血栓的早期精准识别,为血栓形成的可视化预测提供了新策略。Objective To realize the in vivo visual prediction of carotid thrombosis with a thrombin responsive nano-probe in order to provide a novel strategy for the early diagnosis of artery thrombosis.Methods The SPIO was covalently conjugated with Trp and Cy5.5 NHS to construct the probe.The cytotoxicity of the probe was detected with human umbilical vein endothlial cell at different concentrations(0μg/ml,10μg/ml,20μg/ml,50μg/ml,100μg/ml,200μg/ml).Near-infrared fluorescence imaging was used to explore the fluorescence intensity of probes with various thrombin concentrations(0.5 U/ml,1 U/ml,10 U/ml,50 U/ml respectively,blank control group with a thrombin concentration of 0 U/ml and negative control group with a thrombin concentration of 50 U/ml into which 100μg bivalirudin was added).Mice were divided into control or thrombosis group.A carotid thrombosis model of mice in thrombosis group was established.Sections of carotid artery samples were stained with immunofluorescence to analyze the thrombintargeted feature of the probe.Results No significant difference was detected in cell survival rate at different probe concentrations(P>0.05).The fluorescence intensity of the probe at different thrombin concentrations was 17.64-fold,34.24-fold,55.01-fold and 64.65-fold higher than in blank control group(P<0.01).However,there was no significant difference between negative control group and blank control group(P>0.05).The serum thrombin level was significantly higher in thrombosis group than in control group.Conclusion The thrombin responsive probe successfully constructed in this study can realize the early diagnosis of carotid thrombosis accurately,and can thus provide a novel strategy for the in vivo visual prediction of arterial thrombosis.

关 键 词：纳米技术 凝血酶 磁性纳米粒子 血栓形成 荧光共振能量转移 

分 类 号：R543.5[医药卫生—心血管疾病]