摘要
目的制备一种针对整合素αM(CD11b)受体的预定位分子探针^(68)Ga-1,4,7-三氮杂环壬烷-1,4,7-三乙酸-甘氨酸-精氨酸-谷氨酸-精氨酸-谷氨酸-十一聚乙二醇-1,2,4,5-四嗪/CD11b抗体片段-反式-环辛烯[^(68)Ga-NOTA-Polypeptide-PEG11-Tz/anti-CD11b-F(ab′)2-TCO],并通过microPET显像探讨其作为CD11b受体靶向分子探针的可行性。方法采用免疫荧光法检测小鼠巨噬细胞RAW264.7膜表面CD11b受体的表达情况。将CD11b抗体与TCO连接,并通过酶切法得到anti-CD11b-F(ab′)2-TCO。对配体NOTA-Polypeptide-PEG11-Tz进行^(68)Ga标记,检测标记率以及放化纯。进行预定位细胞结合实验,建立CT26结肠癌荷瘤裸鼠模型,进行预定位生物分布以及microPET显像实验。用免疫组织化学检查验证肿瘤微环境中CD11b+细胞的浸润情况。用单因素方差分析比较组间差异。结果免疫荧光检测结果显示RAW264.7细胞膜表面高度表达CD11b受体。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)验证成功合成anti-CD11b-F(ab′)2-TCO。放射性配体^(68)Ga-NOTA-Polypeptide-PEG11-Tz标记率约为94.6%,比活度为7.0~7.4 MBq/μg,放化纯大于95%。预定位细胞结合实验证实该分子探针与CD11b受体有较好的靶向性。生物分布及显像结果示,在预定位4、12以及24 h时间间隔下,模型鼠肾放射性摄取较高,表明分子探针通过肾代谢;肿瘤/肌肉比值为9.23±1.45、12.53±1.36和10.74±1.11(F=848.8,P<0.05);在预定位12 h注射放射性配体后1 h显像,肿瘤与非靶器官对比度最佳:肿瘤标准摄取值(SUV)为0.67±0.12,肌肉SUV为0.09±0.04。免疫组织化学结果示,CT26结肠癌微环境中浸润了大量CD11b+细胞。结论成功合成预定位分子探针^(68)Ga-NOTA-Polypeptide-PEG11-Tz/anti-CD11b-F(ab′)2-TCO,该标记物对CD11b阳性结肠癌具有较强的靶向能力,有望用于靶向CD11b受体的体内示踪。
Objective To prepare a ^(68)Ga labeled probe targeting integrin alpha M(CD11b)receptor,namely ^(68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Glycine-Arginine-Glutamate-Arginine-Glutamate-polyethylene glycol11-1,2,4,5-terazine/CD11b antibody-F(ab′)2-trans-cyclooctene(^(68)Ga-NOTA-Polypeptide-PEG11-Tz/anti-CD11b-F(ab′)2-TCO),and to explore its feasibility as a molecular probe for CD11b receptor through microPET imaging.Methods Immunofluorescence was used to detect the expression of CD11b on the surface of RAW264.7 cell.CD11b specific monoclonal antibody(M1/70)was conjugated with TCO,and anti-CD11b-F(ab′)2-TCO fragment was obtained.The ligand NOTA-Polypeptide-PEG11-Tz was labeled with ^(68)Ga,and its specific activity and radiochemical purity were detected.Pre-targeted cell binding experiment was conducted to evaluate the binding ability of molecular probe.CT26 colon cancer bearing mouse models were established,and then pre-targeted biodistribution and imaging experiments were performed.Immunohistochemical experiment was used to verify the expression of CD11b receptor in tumor.The one-way analysis of variance was used to compare the data.Results The results of immunofluorescence demonstrated CD11b receptor was highly expressed on the surface of RAW264.7 cell.Anti-CD11b-F(ab′)2-TCO fragment was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).^(68)Ga-NOTA-Polypeptide-PEG11-Tz was successfully synthesized,with the labeling efficiency of 94.6%.The specific activity was 7.0-7.4 MBq/μg,and the radiochemical purity was higher than 95%.Pre-targeted cell binding experiment confirmed that the molecular probe bound to the CD11b receptor.The biodistribution and imaging experiments showed that the kidney radioactivity uptake was high at pre-targeted 4,12 and 24 h intervals,which proved that probe was excreted through the urinary system.In addition,molecular probe had higher radioactive uptake at the tumor site,with the tumor/muscle ratios of 9.23±1.45,12.53±1.36 and 10.74±1.11(F=
作者
张颖颖
石岱
徐展
程远
程登峰
Zhang Yinging;Shi Dai;Xu Zhan;Cheng Yuan;Cheng Dengfeng(Shanghai Institute of Medical Imaging Department of Nuclear Medicine,Zhongshan Hospital,Fudan University Nuclear Medicine Institute of Fudan University Cancer Center,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《中华核医学与分子影像杂志》
CAS
CSCD
北大核心
2021年第10期585-591,共7页
Chinese Journal of Nuclear Medicine and Molecular Imaging
基金
国家自然科学基金(11875114)。
