Zhang Shu, Wang Lei, Chen Lu, Xu Huayan, Wu Qiang, Bi Feng, Gao Fabao, Xu Feng
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2015 Oct;32(5):1067-74.
Urokinase plasminogen activator receptor (uPAR) is a membrane protein which is attached to the cellular external membrane. The uPAR expression can be observed both in tumor cells and in tumor-associated stromal cells. Thus, in the present study, the human amino-terminal fragment (hATF), as a targeting element to uPAR, is used to conjugate to the surface of superparamagnetic iron nanoparticle (SPIO). Flowcytometry was used to examine the uPAR expression in different tumor cell lines. The specificity of hATF-SPIO was verified by Prussian blue stain and cell phantom test. The imaging properties of hATF-SPIO were confirmed in vivo magnetic resonance imaging (MRI) of uPAR-elevated colon tumor. Finally, the distribution of hATF-SPIO in tumor tissue was confirmed by pathological staining. Results showed that the three cells in which we screened, presented different expression characteristics, i. e., Hela cells strongly expressed uPAR, HT29 cells moderately expressed uPAR, but Lovo cells didn't express uPAR. In vitro, after incubating with Hela cells, hATF-SPIO could specifically combined to and be subsequently internalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal intensity of Hela cells, after incubation with targeted probe, significantly decreased, and otherwise no obvious changes in Lovo cells both by Prussian blue staining and MRI scans. In vivo, hATF-SPIO could be systematically delivered to HT29 xenograft and accumulated in the tumor tissue which was confirmed by Prussian Blue stain compared to Lovo xenografts. Twenty-four hours after injection of targeting probe, the signal intensity of HT29 xenografts was lower than Lovo ones which was statistically significant. This targeting nanoparticles enabled not only in vitro specifically combining to uPAR positive cells but also in vivo imaging of uPAR moderately elevated colon cancer lesions.
尿激酶型纤溶酶原激活物受体（uPAR）是一种附着于细胞外膜的膜蛋白。在肿瘤细胞和肿瘤相关基质细胞中均可观察到uPAR的表达。因此，在本研究中，人氨基末端片段（hATF）作为uPAR的靶向元件，用于与超顺磁性铁纳米颗粒（SPIO）表面偶联。采用流式细胞术检测不同肿瘤细胞系中uPAR的表达。通过普鲁士蓝染色和细胞模拟试验验证了hATF-SPIO的特异性。在uPAR升高的结肠癌的体内磁共振成像（MRI）中证实了hATF-SPIO的成像特性。最后，通过病理染色证实了hATF-SPIO在肿瘤组织中的分布。结果显示，我们筛选的三种细胞呈现出不同的表达特征，即Hela细胞强烈表达uPAR，HT29细胞中度表达uPAR，但Lovo细胞不表达uPAR。在体外，hATF-SPIO与Hela细胞孵育后，可特异性结合并随后被uPAR阳性细胞内化，这可通过普鲁士蓝染色观察到。同时，与靶向探针孵育后的Hela细胞的T2WI信号强度显著降低，而通过普鲁士蓝染色和MRI扫描，Lovo细胞均无明显变化。在体内，hATF-SPIO可经系统给药至HT29异种移植瘤并在肿瘤组织中蓄积，与Lovo异种移植瘤相比，经普鲁士蓝染色证实。注射靶向探针24小时后，HT29异种移植瘤的信号强度低于Lovo异种移植瘤，差异具有统计学意义。这种靶向纳米颗粒不仅能够在体外特异性结合uPAR阳性细胞，还能够对uPAR中度升高的结肠癌病变进行体内成像。
