协和医学杂志

2013, (03)

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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
The development of a peptide based cancer specific MR contrast agent targeting CXCR4 and validation of its effects in 3 cancer cell lines

朱亮;何泳蓝;金征宇;雷晶;刘健;孟洁;薛华丹;徐海燕

摘要(Abstract):

目的 以恶性肿瘤细胞膜上过量表达的CXCR4为靶点,构建磁共振靶向对比剂,实现三种不同恶性肿瘤细胞的靶向磁共振成像,并探讨磁共振信号与肿瘤细胞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三种恶性肿瘤细胞株(乳腺癌MCF-7、肺癌A549和胰腺癌Panc-1)的CXCR4表达,并验证新型多肽Pep12与三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 cxide nanoparticel, 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;MTS法测定其细胞毒性;MR成像验证不同浓度Pep12-USPIO溶液的T2/T2*信号减低效应。普鲁士蓝染色验证Pep12-USPIO与三种肿瘤细胞的特异性结合,MR成像验证Pep12-USPIO造成与三种肿瘤细胞CXCR4表达量相关的磁共振信号减低的能力。 结果 三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-7:1.8%。多肽Pep12与三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(P=0.01)。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±1.48nm。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.普鲁士蓝染色证实Pep12-USPIO与三种恶性肿瘤细胞实现靶向结合,MR成像证实Pep12-UPSIO能够造成肿瘤细胞悬液磁共振T2/T2*值降低,并且ΔR2/ΔR2*值与肿瘤细胞CXCR4表达水平呈显著的正相关性(前者r=0.997, P=0.050;后者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的表达量呈正相关性。
Objective To construct a cancer specific MR contrast agent targeting the over-expressed CXCR4 on cancer cell surfaces, to testify its effectiveness in binding to cancer cells specifically and producing a decrease in MR T2/T2* signals in vitro, and to discuss its ability to quantify the CXCR4 expression level of various cancer cells. Methods Cellular immunofluorescency experiments and flow cytometry were introduced to observe CXCR4 expression pattern and to quantify CXCR4 expression level in 3 different cancer cell lines( breast cancer cell line MCF-7, lung cancer cell line A549 and pancreatic cancer cell line Panc-1) 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 nanoparticles (USPIO-Np) were synthesized de novo and conjugated to Pep12 after surface modification. Dynamic light scattering(DLS) method was introduced to measure its hydro-diameter, MTS assay to test its cell toxicity, and 1.5T MR scan were carried out to evaluate its T2/T2* signal decrease effect. 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* value change. Results CXCR4 expression were observed in different patterns and levels in all 3 cancer cell lines. Flow cytometry showed the CXCR4 positive cell proportion were 18.7%(PANC-1); 2.9%(A549) and 1.8%(MCF-7), respectively. Pep12 was able to bind to all 3 cancer cell lines specifically, in a CXCR4 level dependent manner. Pep12-USPIO could form stable aqueous colloid in PBS/water under room temperature. The hydro diameter was 86.60±1.48nm nm. Pullulan stain showed pep12-USPIO could bind to PANC-1, A549 and MCF-7 cells, while UPSIO alone could not. PANC-1, A549 and MCF-7 cells were incubated with Pep12-USPIO/UPSIO, and underwent MR scan. A significant T2/T2* signal dropdown was observed in Pep12-USPIO incubated cell suspension, while USPIO incubated cell suspension only had slight T2/T2* signal change.The value of T2(r=0.997, P=0.050)/T2*( r=1.000, P=0.019)change has positive correlation to the expression level of CXCR4 in those tumor cells. Conclusion Pep12-USPIO was stable and hypotoxic, it could specifically bind to CXCR4 expressing cancer cells and produce MR signal change. The value of T2/T2* change might be used for the prediction of CXCR4 expression.

关键词(KeyWords): |恶性肿瘤 分子影像学 磁共振 靶向对比剂 CXCR4|
|cancer molecular imaging MR contrast agent CXCR4|

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作者(Author): 朱亮;何泳蓝;金征宇;雷晶;刘健;孟洁;薛华丹;徐海燕

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