MMP-9单克隆抗体超微超顺磁性氧化铁粒子的合成并行ApoE-/-小鼠动脉粥样硬化7.0 T MR成像的研究  

7.0 T MR detect atherosclerotic plaques in ApoE-/-mice carotid artery by specific immunomagnetic probe

作　　者：姚琼[1] 邓钢[1] 吴国球[2] 张宇[3] 王松[4] 赵瑞[1] 王中娟[1] 居胜红[1] 金晖[5] 

机构地区：[1]东南大学附属中大医院放射科,江苏南京210009 [2]东南大学附属中大医院临床检验中心,江苏南京210009 [3]东南大学生物科学与医学工程系,江苏南京210009 [4]东南大学医学院,江苏南京210009 [5]东南大学附属中大医院内分泌科,江苏南京210009

出　　处：《东南大学学报（医学版）》2011年第3期431-435,共5页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金重点项目(30830039);国家自然科学基金资助项目(30770624)

摘　　要：目的:通过化学合成方法制作基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)单克隆抗体超微超顺磁性氧化铁(ultrasmall superparamagnetic iron oxide,USPIO)粒子MR分子探针,并探讨其作为MR特异性分子探针示踪剂使动脉粥样硬化成像的可行性。方法:采用EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)化学连接剂将MMP-9单克隆抗体与DMSA[meso-2,3-dimercaptosuccinic acid,(HOOCCH(SH)-CH(SH)-COOH)]修饰的USPIO相结合,形成具有免疫学活性的MR分子探针。通过间接酶联免疫吸附试验(ELISA)法检测其免疫活性及生物稳定性。采用高脂饲料喂养ApoE-/-小鼠结合机械损伤的方法,建立小鼠左侧颈动脉粥样硬化模型。将MR磁性粒子探针(Ab-MMP-9-USPIO)经小鼠尾静脉注射(45 mgFe.kg-1),分别于0、4、6、24、48 h对小鼠颈部行7.0 TMR扫描检查。结果:向0.05 mg的USPIO溶液中加入不同质量的单克隆抗体,ELISA试验所得的生成物MMP-9单克隆抗体超微超顺磁性纳米颗粒(Ab-MMP-9-USPIO)吸光度值随抗体含量增加而增大,与阴性对照USPIO组(未加单克隆抗体)相比较差异有统计学意义(P<0.05),分别间隔3 d及100 d后再次测量,100μg及200μg单抗组吸光度值降低(P<0.05)。经小鼠尾静脉注射磁性粒子探针后6 h,损伤侧颈动脉斑块管壁信号于T2WI像上降低最显著,管腔呈"假性扩大样"改变。结论:EDC化学方法可制备出单克隆抗体USPIO粒子,同时仍具有抗体的免疫学活性及生物稳定性。该合成物可以作为MR分子探针在体显像小鼠颈动脉粥样硬化斑块,为动脉粥样硬化斑块的分子成像提供了实验依据。Objective： To synthesize the anti-mouse matrix metalloproteinase-9（MMP-9） immunomagnetic nanoparticles（Ab-MMP-9-USPIO）chemically.Then to investigate the possibility of Ab-MMP-9-USPIO specific probe as a marker of atherosclerotic plaques detected with 7.0 T magnetic resonance imaging（MRI） system in mice models.Methods：Superparamagnetic iron oxide（USPIO） was prepared by chemical coprecipitation and coated with meso-2,3-dimercaptosuccinic acid（HOOCCH（SH）-CH（SH）-COOH） or DMSA].The anti-mouse MMP-9 immunomagnetic nanoparticles（Ab-MMP-9-USPIO） were synthesized by grafting anti-mouse MMP-9 antibodies on the surface of DMSA-coated using the linker of EDC（1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride）.The conjugation amount of the antibodies and the activity of Ab-MMP-9-USPIO were evaluated by enzyme linked immunosorbent assay（ELISA）.Models of atherosclerosis were established by ligating the ApoE-/-mice left carotid artery and feeding with high lipid diet.The mice models were injected Ab-MMP-9-USPIO through tail vein.The MRI was performed at 0,4,6,24,48 h after the injection.Results： The optical density（OD） from ELISA went up with increasing the quality of antibodies.The samples were also measured by ELISA menthod after 3 days and 100 days respectively,and 100,200 μg samples＇ OD descent after 3 days.All the results above were investigated statistically.At 6 hours after the injection of Ab-MMP-9-USPIO into the mice models,the injured vessels showed signal intensity loss on T2WI most obviously.Conclusion：The anti-mouse MMP-9 immunomagnetic nanoparticles can be chemically synthesized by EDC,and the conjugation activity and stability still remain.It is possible to use Ab-MMP-9-USPIO as a molecular probe of detecting atherosclerotic plaques in mice carotid artery with 7.0 T MRI.

关 键 词：基质金属蛋白酶-9 超微超顺磁性氧化铁纳米颗粒 单克隆抗体 核磁共振成像 分子影像学 

分 类 号：R445[医药卫生—影像医学与核医学] R543.5[医药卫生—诊断学]