协和医学杂志

2013, v.4(03) 286-293

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以趋化因子受体4为靶点的恶性肿瘤靶向特异性磁共振对比剂的构建及体外成像
Development of a Peptide-based Cancer-specific Magnetic Resonance Contrast Agent Targeting CXCR4 and Validation of Its Effects in Three Cancer Cell Lines

朱亮;刘健;雷晶;许海燕;孟洁;何泳蓝;金征宇;薛华丹;
ZHU Liang 1 ,LIU Jian 2 ,LEI Jing 2 ,XU Hai-yan 2 ,MENG Jie 2 ,HE Yong-lan 1 , JIN Zheng-yu 1 ,XUE Hua-dan 1 1 Department of Radiology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China 2 Department of Biomedical Engineering,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100005,China

摘要(Abstract):

目的构建以趋化因子受体4(C-X-C motif chemokine receptor4,CXCR4)为靶点的磁共振靶向对比剂并通过体外恶性肿瘤细胞的靶向磁共振成像探讨磁共振信号变化与肿瘤细胞CXCR4表达量的相关性。方法用细胞免疫荧光法和流式细胞计数法分别观察和测定3种恶性肿瘤细胞株(胰腺癌PANC-1、乳腺癌MCF-7和肺癌A549)的CXCR4表达,并验证新型多肽Pep12与3种恶性肿瘤细胞表面的CXCR4特异性结合的能力。化学合成超顺磁性纳米氧化铁颗粒(ultrasmall paramagnetic iron oxide nanoparticle,USPIO-Np),并与Pep12实现共轭联接。动态光散射法测定Pep12-USPIO的水和直径;用MTS细胞增殖与毒性检测试剂盒测定其细胞毒性;磁共振成像检测不同浓度Pep12-USPIO溶液的T2/T2*信号变化。普鲁士蓝染色验证Pep12-USPIO与3种肿瘤细胞的特异性结合,磁共振成像验证Pep12-USPIO所致磁共振信号变化与3种肿瘤细胞CXCR4表达量的相关性。结果 3种恶性肿瘤细胞株均有不同水平的CXCR4表达,流式细胞计数的CXCR4阳性细胞百分比分别为PANC-1:18.7%;A549:2.9%;MCF-7:1.7%。多肽Pep12与3种恶性肿瘤细胞均能特异性结合,并且结合量与CXCR4表达量呈正相关性(r=0.999,P=0.027)。自行合成的Pep12-USPIO在室温下长时间保持稳定的胶体状态,水和直径为(86.60±1.48)nm。Pep12-USPIO细胞毒性较低,铁浓度低于25μg/ml时,ΔR2值和ΔR2*值与铁浓度呈现良好的线性正比关系(△R2:R2=0.996;△R2*:R2=0.977)。普鲁士蓝染色证实Pep12-USPIO能够与3种恶性肿瘤细胞实现靶向结合,磁共振成像证实Pep12-USPIO能够造成肿瘤细胞悬液磁共振T2/T2*值降低(P<0.01),并且ΔR2/ΔR2*值与肿瘤细胞CXCR4表达水平呈显著的正相关(ΔR2:r=0.997,P=0.050;ΔR2*:r=1.000,P=0.019)。结论 Pep12-USPIO物理性质稳定,细胞毒性较低,能够与不同恶性肿瘤细胞株上的CXCR4特异性结合并实现磁共振T2/T2*信号的减低,并且磁共振信号的变化与细胞株CXCR4的表达量呈正相关。
Objective To construct a cancer-specific magnetic resonance contrast agent targeting C-X-C motif chemokine receptor 4 ( CXCR4) on cancer cell surface and to discuss its ability to quantify the CXCR4 expression level of various cancer cells in vitro through the changes of magnetic resonance ( MR) signal. Methods Cellular immunofluorescence and flow cytometry assays were introduced to observe CXCR4 expression pattern and to quantify CXCR4 expression level in 3 different cancer cell lines ( pancreatic cancer cell line PANC-1,breast cancer cell line MCF-7,and lung cancer cell line A549) ,respectively. By replacing the CXCR4 monoclonal antibody with a novel peptide,Pep12,we carried out these experiments again in the same condition,to prove its ability to bind to CXCR4 specifically. Ultrasmall paramagnetic iron oxide nanoparticle ( USPIO-Np) was synthesized de novo and conjugated to Pep12 after surface modification. Dynamic light scattering ( DLS) method was introduced to measure its hydro diameter,MTS assay was performed to test its cell toxicity,and 1. 5T MR scan were carried out to evaluate the T2 / T2* signal changes. Prussian blue staining was introduced to observe the binding pattern of Pep12-USPIO to 3 cancer cell lines,and MR scanning of cells cultured with Pep12-UPSIO were done to evaluate its ability to quantify the CXCR4 expression level on different cancer cells by T2 / T2* signal changes. Results CXCR4 expression was observed in different patterns and levels in all 3 cancer cell lines. Flow cytometry showed that the CXCR4 positive cell proportions were 18. 7% in PANC-1,2. 9% in A549,and 1. 7% in MCF-7,respectively. Pep12 was able to bind to all 3 cancer cell lines specifically in a CXCR4 level dependent manner ( r = 0. 999,P = 0. 027) . Pep12-USPIO formed stable aqueous colloid in PBS / water under room temperature. The hydro diameter was ( 86. 60 ± 1. 48) nm. The cytoxicity of Pep2-USPIO was low. When the concentration of iron was less than 25 μg / ml,the values of △R2 and △R2* were in line with concentration of iron ( △R2: R 2 = 0. 996; △R2* : R 2 = 0. 977) . Prussian blue stain showed pep12-USPIO was bound to PANC-1, A549,and MCF-7 cells,while USPIO alone could not. PANC-1,A549,and MCF-7 cells were incubated with Pep12-USPIO / USPIO,and underwent MR scan. A significant T2 / T2* signal dropdown was observed in Pep12USPIO-incubated cell suspension,while USPIO-incubated cell suspension only had slight T2 / T2* signal change ( P <0. 01) . The value of △R2/△R2* change had positive correlation with the expression level of CXCR4 in those tumor cells ( △R2: r = 0. 997,P = 0. 050; △R2* : r = 1. 000,P = 0. 019) . Conclusions Pep12USPIO is stable and hypotoxic. It can specifically bind to CXCR4-expressing cancer cells and produce MR signal change. The value of T2 / T2* change may be used for the prediction of CXCR4 expression.

关键词(KeyWords): 恶性肿瘤细胞;分子影像学;磁共振靶向对比剂;趋化因子受体4
cancer cell; molecular imaging; magnetic resonance contrast agent; C-X-C motif chemokine receptor 4

Abstract:

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基金项目(Foundation): 国家自然科学基金(30901385);; 北京市科技新星基金(Z111107054511127)

作者(Author): 朱亮;刘健;雷晶;许海燕;孟洁;何泳蓝;金征宇;薛华丹;
ZHU Liang 1 ,LIU Jian 2 ,LEI Jing 2 ,XU Hai-yan 2 ,MENG Jie 2 ,HE Yong-lan 1 , JIN Zheng-yu 1 ,XUE Hua-dan 1 1 Department of Radiology,Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100730,China 2 Department of Biomedical Engineering,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100005,China

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