~99Tc^m-HYNIC-Annexin V的制备及其与多巴胺能神经元凋亡模型的结合特性  

Preparation of ~99Tc^m-Annexin V and in vitro study of its binding characteristics in dopaminergic apoptotic neurons

作　　者：曹卫[1] 黄金莎[2] 孙圣刚[2] 兰晓莉[1] 张永学[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院核医学科,武汉430022 [2]华中科技大学同济医学院附属协和医院神经内科,武汉430022

出　　处：《中华核医学杂志》2006年第5期306-309,共4页Chinese Journal of Nuclear Medicine

基　　金：国家自然科学基金青年基金(30400516); 武汉青年科技晨光计划资助项目(20045006071-2)

摘　　要：目的研究凋亡显像剂^(99)Tc^m-人膜联蛋白V(Annexin V)的制备及其与多巴胺能神经元凋亡模型的体外结合特性。方法运用双功能螯合剂联肼尼克酰胺(HYNIC)进行^(99)Tc^m 标记 Annexin V,形成^(99)Tc^m-HYNIC-Annexin V,经 Sephadex G-25柱层析分离纯化,采用快速薄层层析(ITLC)法检测放化纯。将^(99)Tc^m-HYNIC-Annexin V 与经过1-甲基-4-苯基吡啶离子(MPP^+)处理制模的大鼠肾上腺嗜铬细胞瘤PC12细胞进行体外细胞量及时间-温度结合实验、饱和结合实验和竞争结合实验.得到^(99)Tc^m-HYNIC-Annexin V 与多巴胺能神经元凋亡模型的结合特性,并比较其与正常细胞及不同凋亡水平下细胞模型的结合特性。结果①^(99)Tc^m-YNIC—Annexin V 标记率(64.56±6.23)%,比活度(3.7～74)×10~5kBq/mg,放化纯(93.6±2.48)%,室温放置4 h 后放化纯仍大于90%。②^(99)Tc^m-HYNIC-Annexin V 与多巴胺能神经元凋亡模型的结合具有特异性、可饱和性,Scatchard 图示平衡解离常数(K_d)为(7.16±1.78)nmol/L,最大结合容量(B_(max))为(178.73±32.62)fmol/10~6细胞。结论多巴胺能神经元凋亡模型对^(99)Tc^m-HYNIC-Annexin V 有良好的摄取,可进一步行动物体内研究。Objective The aims of this study were two. One was to find out an optimal method for ^99Tc^m-Annexin V preparation and the other was to investigate the binding characteristics of ^99Tc^m-Annexin V in dopaminergic apoptotic neurons in vitro. Methods For 99Tcm-Annexin V preparation, hydrazine nicotinamide （HYNIC） , a bifunctional chelating agent was used. Product was purified by Sephadex G-25 column chromatography and analyzed with instant thin layer chromatography （ ITLC）. To test the binding characteristics in dopaminergic apoptotic neurons in vitro, a rat pheochromocytoma cell line （PC12） treated with 1-methyl-4-phenylpyridinium （ MPP^＋ ） was used. Tests including time-temperature binding, saturable binding, competition binding between dopaminergic apoptotic neurons and ^99Tc^m-HYNIC-Annexin V and dosedependent MPP^＋ studies were performed and evaluated. Results The labeling rate of 99Tcm was （64.56 ± 6.23 ）%. The specific activity of ^99Tc^m-HYNIC-Annexin V was （3.7 -74） × 10^5 kBq/mg protein. The radiochemical purity was （93.6 ±2.48 ） % and was 〉 90% after 4 hours＇ storage at room temperature. Scatchard plotting suggested that the concentrations of Ka was （7.16 ± 1.78） nmol/L, and B was （ 178.73 ± 32.62） fmol/10^6 cells. Conclusions The preliminary results show that an optimal ^99Tc^m-HYNIC-Annexin V preparation method can be provided. The ^99Tc^m-HYNIC-Annexin V prepared in our laboratory has good receptor-binding activity and may possibly be a potential drug in studying the apoptotic phenomenon in Parkinson＇s disease at early stage in an animal model.

关 键 词：膜联蛋白V 细胞凋亡 帕金森病 多巴胺 神经元 放射配位体测定 

分 类 号：R817[医药卫生—影像医学与核医学]