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转铁蛋白受体(transferrin receptor,TfR)是一类Ⅱ型跨膜糖蛋白,主要参与调控细胞的铁代谢及生长、增殖进程[1]。相关研究结果证实,在癌前病变及侵袭性肿瘤组织中,TfR的表达显著升高,且其表达水平同肿瘤的分级密切相关,如在胰腺癌、结肠癌、肺癌等肿瘤及多种耐药性肿瘤中,TfR的表达明显上调[2-5]。TfR高表达的肿瘤具有恶性程度高、易产生耐药和预后不良等风险。因此,对肿瘤TfR表达水平的在体监测将有助于指导肿瘤患者的临床管理及预后评估。
目前,靶向TfR的分子探针主要包括转铁蛋白及单克隆抗体,其相对分子质量较大,体内循环时间长,易受内源性转铁蛋白竞争的影响,在应用中存在一定的局限性[6]。而小分子多肽因具有相对分子质量小、易于穿透肿瘤组织、稳定性好、血液清除速率快、易于合成及加工修饰、免疫原性低等优势,近年来在肿瘤分子影像学领域表现出良好的应用前景[7]。组氨酸-精氨酸-脯氨酸-酪氨酸-异亮氨酸-丙氨酸-组氨酸(His-Arg-Pro-Tyr-Ile-Ala-His,HRPYIAH,简称T7)是由噬菌体展示技术筛选获得的小分子多肽,能特异性靶向TfR[8-9]。本研究构建了新型靶向TfR的分子探针99Tcm-HRPYIAH(简称99Tcm-T7),并对其理化性质、靶向特性及显像效能进行了初步研究。
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流式细胞术实验结果显示,PANC-1细胞可与TfR单克隆抗体特异性结合,结合率为(98.9±0.1)%,这说明PANC-1细胞表面高表达TfR;而MX-1细胞与TfR单克隆抗体未见明显结合,结合率为(0.2±0.1)%,这表明MX-1细胞表面的TfR呈相对低表达(图1)。
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99Tcm-T7的标记率>95%。在室温下,99Tcm-T7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)%。
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PANC-1细胞与分子探针99Tcm-T7的结合率在孵育后60 min时达到峰值[(16.12±0.01)%],显著高于MX-1细胞[(1.20±0.01)%],且二者间的差异有统计学意义(P<0.001,图2),同流式细胞术测定的TfR表达水平一致。同时,PANC-1阻断组对99Tcm-T7的结合率降低至(2.40±0.01)%,低于PANC-1实验组,且二者间的差异有统计学意义(t=26.91,P<0.001)。
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在PANC-1荷瘤裸鼠模型中,99Tcm-T7可迅速靶向肿瘤部位,注射后30 min肿瘤轮廓显影清晰,较短时间即可获得较高的肿瘤/肌肉比值(2.80±0.22),而MX-1细胞对99Tcm-T7的摄取程度较低,未见明显显影。99Tcm-T7从血液中的清除速率快,主要经肾脏排泄,注射后60 min时除肾脏及膀胱见显像剂分布外,其余组织较少见显像剂滞留(图3)。
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由表1可知,99Tcm-T7经尾静脉注射进入荷瘤裸鼠体内后,除肾脏及肿瘤组织外,其余器官及组织本底均较低,PANC-1细胞对99Tcm-T7的摄取在注射后30 min即达到(0.55±0.18)%ID/g,高于MX-1细胞 [(0.16±0.11)%ID/g],且二者差异有统计学意义(P<0.001)。
器官或组织 人胰腺癌PANC-1细胞 人乳腺癌MX-1细胞 血液 0.34±0.06 0.46±0.16 脑 0.04±0.01 0.04±0.02 心 0.21±0.01 0.12±0.09 肺 0.29±0.01 0.67±0.27 肝 0.34±0.02 1.34±1.70 脾 0.17±0.04 0.10±0.05 肾 5.92±0.04 6.25±0.09 胃 0.36±0.01 0.21±0.09 小肠 0.14±0.04 0.14±0.09 大肠 0.15±0.03 0.13±0.10 肌肉 0.20±0.05 0.10±0.02 骨 0.10±0.02 0.08±0.66 肿瘤 0.55±0.18a 0.16±0.11 注:a表示与人乳腺癌PANC-1细胞比较,差异有统计学意义(t=6.42,P=0.003)。T7为组氨酸-精氨酸-脯氨酸-酪氨酸-异亮氨酸-丙氨酸-组氨酸;%ID/g为每克组织百分注射剂量率 表 1 荷人胰腺癌、人乳腺癌裸鼠注射分子探针99Tcm-T7后30 min的体内生物学分布(%ID/g,n=5,
)$\bar x \pm s $ Table 1. Biodistribution of molecular probe 99Tcm-T7 in nude mice bearing human pancreatic cancer and human breast cancer at 30 min post injection (%ID/g, n=5,
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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(图4)。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(图5)。
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
Construction of a transferrin receptor targeting probe 99Tcm-T7 for noninvasive imaging of tumor
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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。 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. -
表 1 荷人胰腺癌、人乳腺癌裸鼠注射分子探针99Tcm-T7后30 min的体内生物学分布(%ID/g,n=5,
)$\bar x \pm s $ Table 1. Biodistribution of molecular probe 99Tcm-T7 in nude mice bearing human pancreatic cancer and human breast cancer at 30 min post injection (%ID/g, n=5,
)$\bar x \pm s $ 器官或组织 人胰腺癌PANC-1细胞 人乳腺癌MX-1细胞 血液 0.34±0.06 0.46±0.16 脑 0.04±0.01 0.04±0.02 心 0.21±0.01 0.12±0.09 肺 0.29±0.01 0.67±0.27 肝 0.34±0.02 1.34±1.70 脾 0.17±0.04 0.10±0.05 肾 5.92±0.04 6.25±0.09 胃 0.36±0.01 0.21±0.09 小肠 0.14±0.04 0.14±0.09 大肠 0.15±0.03 0.13±0.10 肌肉 0.20±0.05 0.10±0.02 骨 0.10±0.02 0.08±0.66 肿瘤 0.55±0.18a 0.16±0.11 注:a表示与人乳腺癌PANC-1细胞比较,差异有统计学意义(t=6.42,P=0.003)。T7为组氨酸-精氨酸-脯氨酸-酪氨酸-异亮氨酸-丙氨酸-组氨酸;%ID/g为每克组织百分注射剂量率 -
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