| 转铁蛋白受体靶向分子探针99Tcm-T7的放射性标记及肿瘤显像研究 |
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| 引用本文： | 肖晴,潘芯,李崇佼,蒋亚群,王怡春,文兵,雷萍,何勇. 转铁蛋白受体靶向分子探针99Tcm-T7的放射性标记及肿瘤显像研究[J]. 国际放射医学核医学杂志, 2022, 46(4): 223-229. DOI: 10.3760/cma.j.cn121381-202110017-00167 |
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| 作者姓名： | 肖晴 潘芯 李崇佼 蒋亚群 王怡春 文兵 雷萍 何勇 |
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| 作者单位： | 1.武汉大学中南医院核医学科，武汉 430071 |
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| 摘 要： | 目的 制备靶向转铁蛋白受体（TfR）的多肽分子探针99Tcm-组氨酸-精氨酸-脯氨酸-酪氨酸-异亮氨酸-丙氨酸-组氨酸（简称99Tcm-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99Tcm-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99Tcm-T7体外结合TfR的亲合力及特异性。构建荷瘤裸鼠模型，注射99Tcm-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99Tcm-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99Tcm-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99Tcm-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，99Tcm-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99Tcm-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99Tcm-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99Tcm-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]，差异有统计学意义（t=6.42，P<0.001）。放射性磷屏自显影结果显示，在注射99Tcm-T7 30 min后，相较于MX-1细胞，PANC-1细胞显著摄取99Tcm-T7；在正常组织脏器中，以肾脏摄取最为显著，其次为肝脏。苏木精-伊红染色法及免疫组织化学染色法结果显示，肿瘤实质内未见明显坏死，在PANC-1细胞中TfR呈高表达，而在MX-1细胞中TfR呈低表达。 结论 成功制备了靶向TfR的特异性多肽分子探针99Tcm-T7，其在荷瘤裸鼠模型中具有良好的显像效能，有望为在体监测肿瘤TfR的表达提供新的影像学手段。
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| 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 |
| 收稿时间： | 2021-10-15 |
Construction of a transferrin receptor targeting probe 99Tcm-T7 for noninvasive imaging of tumor |
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| Qing Xiao,Xin Pan,Chongjiao Li,Yaqun Jiang,Yichun Wang,Bing Wen,Ping Lei,Yong He. Construction of a transferrin receptor targeting probe 99Tcm-T7 for noninvasive imaging of tumor[J]. International Journal of Radiation Medicine and Nuclear Medicine, 2022, 46(4): 223-229. DOI: 10.3760/cma.j.cn121381-202110017-00167 |
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| Authors: | Qing Xiao Xin Pan Chongjiao Li Yaqun Jiang Yichun Wang Bing Wen Ping Lei Yong He |
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| Affiliation: | 1.Department of Nuclear Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, China |
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| Abstract: | Objective To develop a radiolabeled peptide molecular tracer 99Tcm-His-Arg-Pro-Tyr-Ile-Ala-His (99Tcm-T7) targeting transferrin receptor and evaluate its micro SPECT/CT imaging effect in tumor-bearing nude mice models. Methods The peptide probe 99Tcm-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 99Tcm-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 99Tcm-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 99Tcm-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 99Tcm-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 of PANC-1 blocking group to 99Tcm-T7 decreased to (2.40±0.01)%, which was significantly different from that of PANC-1 experimental group(t=26.91, P<0.001). The results of micro SPECT/CT imaging in nude mice bearing tumor showed that 99Tcm-T7 could be quickly cleared from the blood and mainly eliminated from the kidneys. PANC-1 tumor-bearing nude mice models showed clear tumor contour 30 minutes after injection of 99Tcm-T7, with a tumor-to-muscle ratio of 2.80±0.22. The results of biological distribution experiments showed that the uptake of 99Tcm-T7 by tumors and organs (percentage injection dose rate (%ID/g) per gram of tissue) was consistent with the imaging results, and the uptake of 99Tcm-T7 in PANC-1 cells ((0.55±0.18)%ID/g) was higher than that in MX-1 cells ((0.16±0.11)%ID/g), and the difference was statistically significant (t=6.42, P<0.001). The radio-autograph assay showed that PANC-1 cells significantly absorbed 99Tcm-T7 compared with MX-1 cells 30 minutes after injection of 99Tcm-T7. The highest uptake in normal organs was observed in the kidney, followed by the liver. Hematoxylin-eosin and immunohistochemical staining revealed no obvious necrosis in the tumor parenchyma. The PANC-1 cells overexpressed TfR, and whereas the MX-1 cells had low TfR expression. Conclusion A specific polypeptide molecular probe 99Tcm-T7 targeting TfR was successfully prepared, which has excellent imaging efficiency in tumor-bearing nude mice models, and is expected to provide a new imaging method for monitoring the expression of tumor TFR in vivo. |
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