靶向Rho激酶1纳米探针联合MRI可视化动脉粥样硬化斑块的实验研究  

Experimental MRI study of targeting Rho-associated protein kinase 1 to detect plaques in atherosclerosis

作　　者：杨雅雯 夏敏 宋梦星 马占龙 YANG Yawen;XIA Min;SONG Mengxing;MA Zhanlong(Department of Radiology,the First Affiliated Hospital of Nanjing Medical University(People's Hospital of Jiangsu Province),Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院(江苏省人民医院)放射科,南京210029

出　　处：《磁共振成像》2024年第4期106-112,共7页Chinese Journal of Magnetic Resonance Imaging

基　　金：国家自然科学基金项目(编号:81971669)。

摘　　要：目的评估Rho激酶(Rho-kinase,ROCK)1在动脉粥样硬化(atherosclerosis,AS)斑块中的表达,合成ROCK1靶向探针,并探索其可视化AS斑块的可行性。材料与方法ROCK1抗体与超小超顺磁性氧化铁纳米颗粒偶联制备靶向探针(Fe3O4@PEG-ROCK1)并表征。高脂喂养载脂蛋白E基因敲除(Apolipoprotein-Edeficient,ApoE^(-/-))小鼠,在10、16、22、28、34周随机取小鼠(n=5)测量体质量。主动脉免疫染色切片及蛋白印迹实验观察ROCK1的表达及活性。将34周ApoE^(-/-)小鼠分成两组,一组尾静脉注射Fe3O4@PEG(n=10),一组尾静脉注射Fe3O4@PEG-ROCK1(n=10),注射探针前及注射后8、16 h进行MRI。Image J软件计算斑块信号。病理分析腹主动脉标本。结果Fe3O4@PEG和Fe3O4@PEG-ROCK1在水溶液中均匀分散,水合粒径分别为(27.06±1.52)nm及(30.52±2.95)nm,Zeta电位分别为(−35.18±0.31)mV及(−16.60±3.26)mV。Fe3O4@PEG-ROCK1可降低巨噬细胞的吞噬清除,在一定浓度范围内无毒,且保持免疫活性。Fe3O4@PEG-ROCK1饱和磁化强度为0.0868 T及T2弛豫率为162.3 mM^(-1 )s^(-1)说明探针磁敏感性较好。随着AS进展,ApoE^(-/-)鼠体质量明显增加,ROCK1在斑块中的表达逐渐增多(r=0.959,P<0.001)。ApoE^(-/-)鼠腹主动脉ROCK1活性高于健康C57BL/6鼠(0.30±0.02 vs.0.24±0.02,P<0.001)。平扫时Fe3O4@PEG和Fe3O4@PEG-ROCK1组斑块信号分别为8.25±1.39和7.81±3.22,差异无统计学意义;注射探针后两组斑块信号均减低,与Fe3O4@PEG组相比,Fe3O4@PEG-ROCK1组的斑块信号减低更明显(8 h,5.37±1.79 vs.3.91±2.26,P=0.001;16 h,6.68±2.39 vs.4.61±2.80,P=0.001)。普鲁士蓝染色显示的铁纳米沉积区域与免疫组化的ROCK1阳性区域对应。结论ROCK1在AS斑块中表达高和活性高。ROCK1靶向探针(Fe3O4@PEG-ROCK1)是有效磁共振对比剂,有助于实现风险斑块的无创监测。Objective:To observe the expression of Rho-kinase(ROCK)1 in atherosclerotic plaques,to synthesize a ROCK1-targeted probe and characterize it,then explore its ability to visualize atherosclerotic plaques.Materials and Methods:The ROCK1 antibody was coupled with ultra-small superparamagnetic iron oxide nanoparticles to prepare the targeting probe(Fe3O4@PEG-ROCK1),which was characterized and analyzed.Apolipoprotein E knockout(ApoE^(-/-))mice were fed with high-fat diet and five mice were selected randomly at 10,16,22,28,and 34 weeks,respectively,to measure weight.The expression and activity of ROCK1 were observed by ROCK1 immunostaining and western blot.ApoE^(-/-)mice fed for 34 weeks were divided into two groups,one group(n=10)was injected with Fe3O4@PEG,the other group(n=10)was injected with [e-mail] resonance imaging was performed before and 8 h and 16 h after injection of the nanoprobe,and the signal intensity of plaques was calculated by Image J software.Abdominal aortic specimens were analyzed by pathology.Results:Fe3O4@PEG and Fe3O4@PEG-ROCK1 were uniformly dispersed in aqueous solution,and the hydrated particle sizes were(27.06±1.52)nm and(30.52±2.95)nm,respectively.The Zeta potential was(−35.18±0.31)mV and(−16.60±3.26)mV,respectively.Fe3O4@PEG-ROCK1 could reduce the phagocytosis and clearance of macrophages,was non-toxic within a certain concentration range,and maintains immune activity.The saturation magnetization(0.0868 T)and T2 relaxation rate(162.3 mM^(-1) s^(-1))indicated that Fe3O4@PEG-ROCK1 had good magnetic sensitivity.With advancing atherosclerosis,the expression of ROCK1 increased(r=0.959,P<0.001).The activity of ROCK1 in abdominal aorta of ApoE^(-/-)mice was higher than that of C57BL/6 mice(0.30±0.02 vs.0.24±0.02,P<0.01).The results of magnetic resonance imaging showed that compared with plain scan(the plaque signal of Fe3O4@PEG group and Fe3O4@PEG-ROCK1 group were 8.25±1.39 and 7.81±3.22,respectively).After the injection of the probe,the plaque signal of the two group

关 键 词：小鼠 动脉粥样硬化 RHO激酶 纳米探针 分子影像 磁共振成像 

分 类 号：R445.2[医药卫生—影像医学与核医学] R-332[医药卫生—诊断学]