CAJ | 학술논문

目的探讨99Tcm标记人表皮生长因子受体2(HER2)小分子靶向结合蛋白ZHER2:342的制备方法,分析其在体外与HER2的结合特性.方法利用配体交换法进行ZHER1:342的99Tcm标记,分析不同质量的SnC12和NaOH、不同反应时间对标记率的影响,探讨最佳的标记方法.用HPLC测定标记物的标记率与放化纯,并分析其体外稳定性.分析标记物在体外与HER2高表达的人卵巢癌SKOV-3细胞的结合率及滞留率,并与HER2低表达的人乳腺癌MDA-MB-231细胞进行对比.在体外用过量未标记的ZHER2:342对SKOV-3细胞HER2进行封闭后,与未封闭组进行对比,研究该分子探针与HER2结合的特异性.采用单因素方差分析及两样本t检验分析数据.结果 99Tcm标记ZHER2:342的最佳条件为:反应体系中依次加入ZHER2:342 5μg(1 g/L)、NaOH 5 μg(1 g/L)、SnCl2 8.8μg(1 g/L)、99TcmO:150μl(37 MBq),轻微振荡后室温静置1h.99Tcm-ZHER2:342标记率为(98.10±1.73)％,放化纯＞98％;体外稳定性好,与生理盐水及新鲜人血清混合24h后放化纯均＞85％.在体外与HER2高表达的SKOV-3细胞具有较高的结合率,混合后6h最高,结合率为(9.95±1.02)％,且各时间点细胞结合率均明显高于HER2低表达的MDA-MB-231细胞(5.68～9.88与0.56～2.11;t=-34.50～-13.14,均P＜0.01).此外,加入过量的未标记的ZHER2:342进行受体封闭时,99Tcm-ZHER2:342与SKOV-3细胞的结合率从(9.95±1.02)％降到(2.11±0.27)％(t=-13.14,P＜0.01).结论 99Tcm标记ZHER2:342方法简单,标记率高;标记产物体外稳定性好,在体外可与HER2高表达的人卵巢癌SKOV-3细胞特异性结合,是有潜力的HER2靶向分子影像探针.
목적 탐토99Tcm표기인표피생장인자수체2(HER2)소분자파향결합단백ZHER2:342적제비방법,분석기재체외여HER2적결합특성.방법 이용배체교환법진행ZHER1:342적99Tcm표기,분석불동질량적SnC12화NaOH、불동반응시간대표기솔적영향,탐토최가적표기방법.용HPLC측정표기물적표기솔여방화순,병분석기체외은정성.분석표기물재체외여HER2고표체적인란소암SKOV-3세포적결합솔급체류솔,병여HER2저표체적인유선암MDA-MB-231세포진행대비.재체외용과량미표기적ZHER2:342대SKOV-3세포HER2진행봉폐후,여미봉폐조진행대비,연구해분자탐침여HER2결합적특이성.채용단인소방차분석급량양본t검험분석수거.결과 99Tcm표기ZHER2:342적최가조건위:반응체계중의차가입ZHER2:342 5μg(1 g/L)、NaOH 5 μg(1 g/L)、SnCl2 8.8μg(1 g/L)、99TcmO:150μl(37 MBq),경미진탕후실온정치1h.99Tcm-ZHER2:342표기솔위(98.10±1.73)％,방화순＞98％;체외은정성호,여생리염수급신선인혈청혼합24h후방화순균＞85％.재체외여HER2고표체적SKOV-3세포구유교고적결합솔,혼합후6h최고,결합솔위(9.95±1.02)％,차각시간점세포결합솔균명현고우HER2저표체적MDA-MB-231세포(5.68～9.88여0.56～2.11;t=-34.50～-13.14,균P＜0.01).차외,가입과량적미표기적ZHER2:342진행수체봉폐시,99Tcm-ZHER2:342여SKOV-3세포적결합솔종(9.95±1.02)％강도(2.11±0.27)％(t=-13.14,P＜0.01).결론 99Tcm표기ZHER2:342방법간단,표기솔고;표기산물체외은정성호,재체외가여HER2고표체적인란소암SKOV-3세포특이성결합,시유잠력적HER2파향분자영상탐침.
Objective To prepare the 99Tcm-labeled human epidermal growth factor receptor type 2 (HER2) affibody molecule ZHER2:342 and evaluate its receptor binding specificity in vitro.Methods The molecular ZHERa:342 was labeled with 99Tcm using the ligand exchange method.The labeling efficiency and radiochemical purity were measured by HPLC.The major factors,such as the mass of SnC12 and NaOH and reaction time were analyzed,and the optimal method was summarized.Cell binding kinetics and cellular retention of the probe were investigated in HER2-expressing SKOV-3 cells and MDA-MB-231 cells with low HER2 expression respectively.HER2 binding specificity of 99Tcm-ZHER2:342 was analyzed by a pre-injection of excess unlabeled ZHER2:342 to saturate HER2 receptors.One-way analysis of variance and two-sample t test were used.Results The optimal labeling procedure was as follows:5 μg (1 g/L) of ZHER2:342 was mixed with 5 μg of NaOH (1 g/L),then 8.8 μg SnC12(1 g/L,solution) was added,followed by 150 μl (37 MBq) 99TcmO4-solution,and finally the mixture was slightly vortexed and incubated for 1 h at room temperature.99TcmZHER2:342 was stable in vitro with a high labeling efficiency of (98.10± 1.73)％.The radiochemical purity was ＞ 98％,and was more than 85％ after the incubation for 24 h in saline and fresh human serum.The cell binding of 99Tcm-ZHER2:342 with HER2-expressing SKOV-3 cells gradually increased over time with a peak of (9.95± 1.02)％ at 6 h.The binding of 99Tcm-ZHER2:342 in SKOV-3 cells was significantly higher than that in MDA-MB-231 cells at every time point (5.68-9.88 vs 0.56-2.11 ; t:from-34.50 to-13.14,all P＜0.01).The labeled molecular probe retained the capacity to bind specifically to HER2-expressing SKOV-3 cells since the cell binding decreased from (9.95 ± 1.02) ％ to (2.11 ±0.27) ％ after receptor saturation (t =-13.14,P＜0.01).Conclusions 99Tcm-ZHER2:342 has a high labeling efficiency,good stability and optimal binding specificity.These characteristics enable it to be a promising molecular probe for HER2-targeting imaging.