转铁蛋白受体靶向分子探针^(99)Tc^(m)-T7的放射性标记及肿瘤显像研究  

Construction of a transferrin receptor targeting probe^(99)Tc^(m)-T7 for noninvasive imaging of tumor

作　　者：肖晴 潘芯 李崇佼 蒋亚群 王怡春 文兵[1] 雷萍[2] 何勇 Xiao Qing;Pan Xin;Li Chongjiao;Jiang Yaqun;Wang Yichun;Wen Bing;Lei Ping;He Yong(Department of Nuclear Medicine,Zhongnan Hospital of Wuhan University,Wuhan 430071,China;Department of Immunology,School of Basic Medical Science,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]武汉大学中南医院核医学科,武汉430071 [2]华中科技大学同济医学院基础医学院免疫学系,武汉430030

出　　处：《国际放射医学核医学杂志》2022年第4期223-229,共7页International Journal of Radiation Medicine and Nuclear Medicine

基　　金：国家自然科学基金(81871391);国家疑难病症诊治能力提升工程建设项目肿瘤方向(ZLYNXM202007);武汉大学中南医院医学科技创新平台支撑项目(PTXM2020014)。

摘　　要：目的制备靶向转铁蛋白受体(TfR)的多肽分子探针^(99)Tc^(m)-组氨酸-精氨酸-脯氨酸-酪氨酸-异亮氨酸-丙氨酸-组氨酸(简称^(99)Tc^(m)-T7),并评估其在荷瘤裸鼠模型micro SPECT/CT显像中的效果。方法采用间接标记法,在共配体N-三(羟甲基)甲基甘氨酸和乙二胺二乙酸的协同下,制备^(99)Tc^(m)-T7。采用流式细胞术测定人胰腺癌PANC-1细胞和人乳腺癌MX-1细胞表面TfR的表达水平,采用体外细胞结合及细胞竞争抑制实验评估^(99)Tc^(m)-T7体外结合TfR的亲合力及特异性。构建荷瘤裸鼠模型,注射^(99)Tc^(m)-T7后进行micro SPECT/CT显像及生物学分布实验。采用离体组织放射性磷屏自显影成像及组织病理学检查,观察TfR的表达情况。2组间的比较采用独立样本t检验。结果成功制备了分子探针^(99)Tc^(m)-T7,其标记率>95%,分别在与生理盐水、胎牛血清的混合液中孵育4 h后的放射化学纯度为(95.3±0.3)%和(90.6±0.4)%。流式细胞术实验结果显示,PANC-1细胞与TfR单克隆抗体的结合率为(98.9±0.1)%,而MX-1细胞与TfR单克隆抗体的结合率为(0.2±0.1)%。体外细胞结合实验结果表明,PANC-1细胞与^(99)Tc^(m)-T7共孵育60 min后结合率达到峰值[(16.12±0.01)%],高于MX-1细胞[(1.20±0.01)%],且二者间的差异有统计学意义(t=28.67,P<0.001);细胞竞争抑制实验结果表明,PANC-1阻断组与^(99)Tc^(m)-T7的结合率降低至(2.40±0.01)%,与PANC-1实验组的差异有统计学意义(t=26.91,P<0.001)。荷瘤裸鼠体内micro SPECT/CT显像结果显示,^(99)Tc^(m)-T7可从血液中迅速清除,主要通过肾脏排泄。PANC-1荷瘤裸鼠模型在注射^(99)Tc^(m)-T7后30 min时肿瘤轮廓显示清晰,肿瘤/肌肉比值达2.80±0.22。生物学分布实验结果显示,肿瘤及各脏器对^(99)Tc^(m)-T7的摄取[每克组织百分注射剂量率(%ID/g)]与显像结果一致,且^(99)Tc^(m)-T7在PANC-1细胞中的摄取[(0.55±0.18)%ID/g]高于MX-1细胞[(0.16±0.11)%ID/g],差异有统计学意Objective To develop a radiolabeled peptide molecular tracer^(99)Tc^(m)-His-Arg-Pro-Tyr-Ile-Ala-His(^(99)Tc^(m)-T7)targeting transferrin receptor and evaluate its micro SPECT/CT imaging effect in tumor-bearing nude mice models.Methods The peptide probe^(99)Tc^(m)-T7 was developed by indirect labeling method with the coordination of co-ligands N-tri(hydroxymethyl)methylglycine and ethylenediamine diacetate.The expression levels of TfR on the surface of human pancreatic PANC-1 tumor cells and human breast MX-1 tumor cells were measured through flow cytometry.Cell binding and competitive blocking assays was conducted to analyze the binding affinity and specificity of^(99)Tc^(m)-T7 in vitro.Micro SPECT/CT imaging and biodistribution after the establishment of mouse xenograft models were performed in vivo to evaluate the affinity and feasibility of noninvasive tumor imaging.Radio-autograph assay and immunohistochemical staining were conducted to validate the correlation between the uptake of^(99)Tc^(m)-T7 and expression of TfR in tumor tissues.Independent sample t-test was used for the comparison between the two groups.Results The radiolabeled probe^(99)Tc^(m)-T7 was successfully synthesized with a radiolabeling yield of greater than 95%.It exhibited great stability in vitro,with radiochemical purities of(95.3±0.3)%and(90.6±0.4)%after incubation in normal saline and fetal bovine serum for 4 hours,respectively.The results of flow cytometry showed that PANC-1 tumor cells overexpressed TfR on the surface with a high tendency to bind TfR monoclonal antibody((98.9±0.1)%),whereas MX-1 tumor cells showed low TfR expression on the membrane((0.2±0.1)%).In vitro cell binding assay results showed that the binding rate of PANC-1 cells to^(99)Tc^(m)-T7 reached a peak((16.12±0.01)%)after 60 minutes of incubation,which was higher than that of MX-1 cells((1.20±0.01)%),and the difference between them was statistically significant(t=28.67,P<0.001).The results of cell competition inhibition experiment showed that the binding rate

关 键 词：受体 转铁蛋白 肽类 分子探针 锝 同位素标记 放射免疫显像 单光子发射计算机体层摄影术 肿瘤细胞 培养的 小鼠 裸 

分 类 号：R730.44[医药卫生—肿瘤]