^(99m)Tc-MMP-9Ab无创探测ApoE-/-小鼠颈动脉粥样硬化斑块

Noninvasive in vivo detection of atherosclerotic plaques by ^(99m)Tc labeled matrix metalloproteinase-9 antibody in ApoE-/-mice

目的建立载脂蛋白E基因敲除(ApoE-/-)小鼠颈动脉动脉粥样硬化(AS)模型,探讨99mTc标记基质金属蛋白酶-9抗体(MMP-9Ab)标记的最佳条件以及用99mTc-MMP-9Ab无创性探测AS斑块的可行性。方法损伤并阻断10只ApoE-/-小鼠左侧颈总动脉血流20 min后高脂饮食饲养4周。氯化亚锡晶体(SnCl2.2H2O)的用量为31.25～1000.00μg,MMP-9Ab的用量为0.1、0.2、0.3ml,测定不同条件下标记物的标记率及其稳定性。将适量标记物经小鼠尾静脉注射,于注射后不同时间点取主要脏器测量放射性计数率值,经放射性衰变校正后计算每克组织的百分注射剂量率(%ID/g),观察99mTc-MMP-9Ab在正常小鼠体内的生物分布情况。对ApoE-/-AS模型鼠及C57BL/6小鼠各5只行左侧颈总动脉离体自显影显像,观察99mTc-MMP-9Ab在ApoE-/-小鼠动脉粥样硬化模型中体外的显像情况。结果 99mTc-MMP-9Ab标记的最佳条件为SnCl2.2H2O溶液(10g/L)0.05ml,柠檬酸溶液0.1ml,MMP-9Ab溶液(0.2g/L)0.1ml,通氮气振荡1h,再加入新鲜99mTcO4-185MBq,100℃水浴30min,99mTc-MMP-9Ab标记率可达(95.16±1.81)%,室温下放置3h后放化纯不小于80%。99mTc-MMP-9Ab在正常小鼠血液内清除迅速,30min后血液中放射性迅速下降,肌肉内放射性亦甚少。离体自显影显像中,粥样硬化组自显影胶片上的曝光黑影与肉眼可见的粥样硬化斑块有一致性。结论本研究建立的99mTc标记MMP-9Ab方法操作简便,反应时间短,标记率高,体外稳定性较好,无需进一步纯化。99mTc-MMP-9Ab有望成为显示AS斑块形成的靶向显像剂。

Objective To establish atherosclerosis（AS） models using Apolipoprotein E-deficient（ApoE-/-） mice,and to observe the optimal labeling conditions for 99mTc labeled matrix metalloproteinase-9 antibody（99mTc-MMP-9Ab）,as well as the possibility for imaging experimental AS plaques in ApoE-/-mice.Methods Ten ApoE-/-mice were fed with high lipid diet for 4 weeks after injury of the left carotid arteries and the blood interception for 20 min.The amount of stannous chloride（SnCl2·2H2O） changed from 31.25 to 1000 μg,of MMP-9Ab was 0.1,0.2 and 0.3 ml.The labeling efficiency and the in vitro stability of 99mTc-MMP-9Ab were analyzed.The normal mice were killed,and the major organs were taken out from those at 5,30,60 and 120 min after 99mTc-MMP-9Ab injection through the tail veins,the radioactivity count of those organs were measured.After 0.1 ml 99mTc-MMP-9Ab was injected into tail veins of 5 ApoE-/-AS mice and 5 C57BL/6 mice,the mice were sacrificed,their left common carotid arteries were removed and covered with X-ray films.The films were developed and fixed after exposing for 1 h in refrigerator.Results The optimal conditions for 99mTc-MMP-9Ab labeling reaction were as follows： 0.05 ml SnCl2·2H2O（10 g/L） dissolved by 0.1 ml citric acid solution,MMP-9Ab（0.2 g/L） 0.1 ml mixed at room temperature for 1 h with nitrogen atmosphere and continuous agitating,0.1 ml fresh 99mTcO-4（185 MBq） added,water bathing 100℃ for 30 min.Biodistribution studies revealed the clearance of 99mTc-MMP-9Ab from blood was rapid.On the radioautography films,shadows of AS plaques were clearly visible.Conclusion This method of labeling MMP-9Ab with 99mTc is convenient with satisfactory in vitro stability and labeling efficiency,and no further purification needed to the labeling radiopharmaceutical before administration.99mTc-MMP-9Ab has the potential for rapid noninvasive detection of plaque formation.