^(99)Tc^m-FM2心肌细胞凋亡显像的实验研究

Experimental study of myocardial apoptosis with ^(99)Tc^m-FM2 imaging

目的探讨(99)~Tc^m 标记突触结合蛋白Ⅰ的 C2A 片段-谷胱甘肽转移酶复合物(FM2)在心肌细胞凋亡显像中的应用价值。方法通过基因工程合成 FM2,以2-亚氨基噻吩盐酸盐(IT)修饰,经葡庚糖酸钠(GH)转换标记制备(99)~Tc^m-FM2。(99)~Tc^m-FM2经纯化后,纸层析法测定放化纯。破碎红细胞膜磷脂结合实验检测(99)~Tc^m-FM2在 Ca^(2+)介导下与凋亡细胞表面磷脂结合的能力。以6头小型猪制备心肌凋亡模型,通过心导管将球囊送入并充气阻塞冠状动脉主要分支远端(左前降支、左回旋支或右冠状动脉),20～30 min 后,撤除球囊,造成缺血再灌注损伤,引发急性心肌梗死。注射(99)~Tc^m-FM2后3 h,进行 CT 定位和 SPECT 显像,计算心肌异常高放射性区/本底(T/B)比值。处死动物,体外测定凋亡心肌与正常心肌的单位质量放射性比值。用流式细胞分析和电镜检查验证显像结果。结果 (99)~Tc^m-FM2稳定,放化纯>95%。FM2经(99)~Tc^m 标记后仍保持 Ca^(2+)介导的与膜磷脂特异结合的能力。活体显像:(99)~Tc^m-FM2注射后3 h,凋亡心肌清晰显影,T/B 比值为3.36±0.74。体外测定:缺血再灌注损伤心肌与正常心肌的单位质量放射性比值为11.68±4.02。流式细胞分析和电镜检查均证实心肌高放射性区凋亡心肌组织形成。结论 (99)~Tc^m-FM2在活体内与凋亡心肌组织特异结合,对无创性检测心肌细胞凋亡有潜在应用价值。

Objective To evaluate the value of ^99Tc^m-C2A domain of synaptotagmin I -glutathione-S-transferase fusion protein （FM2） for the non-invasive assessment of myocardial apoptosis using SPECT. Methods Recombinant C2A domain of synaptotagmin I was overexpressed in E. Coli, and purified as glutathione-S-transferase, FM2 was produced through this process. FM2 was thiolated with 2-iminothiolane（2-IT） and labeled with ^99Tc^m. Radiochemical purity was determined with thin layer chromatogra- phy. To ensure that the binding activity of ^99Tc^m-FM2 and Ca^2 ＋ -dependent phosphatidylserine （PS） was retained, ^99Tc^m-FM2 was tested by using lyses blood cell membranes in both presence and absence of Ca^2＋. The radiotracer was tested with ischemia-reperfusion models in 6 pigs. Briefly, occlusion of the left descending coronary artery or circumflex branch or right coronary artery of adult pigs was induced by balloon angioplasty for 20 - 30 min, then followed by reperfusion, ^99Tc^m-FM2 was intravenously injected and SPECT images were acquired at 3 h post-injection. Results ^99Tc^m-FM2 had good radiochemical purity （ 〉 95% ） ; and the binding activity of ^99Tc^m-FM2 and Ca^2＋ -dependent PS was well preserved, There was local uptake of radioactivity at the experimental infarct areas, with target/background （T/B） ratio being 3.36 ±0. 74 in pigs at 3 h post-injection of the radiotracer using SPECT, while T/B ratio was 11.68 ±4.02 in direct counting of the infarct tissue and normal myocardium. Myocardial apoptosis in infarct areas was confirmed by flow cytometry and electron microscopy. Conclusions^99Tc^m-FM2 binds to apoptotic cells by recognizing exposed PS as a molecular marker. It should be a potential radiotracer for imaging of myocardial apoptosis.