标记hATF的超顺磁性氧化铁纳米颗粒探针在结肠癌肿瘤模型中的分子影像

Molecular Image of Superparamagnetic Iron Oxide Nanopariticle Labeled with hATF in Colon Tumor Models

尿激酶型纤溶酶原激活物受体(uPAR)是细胞外膜蛋白。uPAR不仅在恶性肿瘤细胞中表达明显增高,同时在肿瘤间质中也表达增强。本研究将uPAR作为分子靶点,以人类氨基末端片段(hATF)作为靶向原件标记超顺磁性氧化铁纳米颗粒(SPIO),利用该靶向探针在uPAR中度表达的HT29荷瘤裸鼠体内验证该探针的影像效能。体外通过流式细胞术检测细胞uPAR表达量,普鲁士蓝染色以及细胞MRI扫描检测探针的特异性,MRI扫描荷瘤裸鼠检测探针体内的成像效能,并通过病理学检查显示探针在肿瘤内分布情况。结果显示,不同肿瘤细胞具有不同uPAR表达特性,在筛选的细胞系中,Hela为uPAR强阳性细胞,HT29为uPAR中度阳性细胞,Lovo则为uPAR阴性细胞。体外试验利用强表达uPAR的Hela细胞与hATF-SPIO探针共同孵育,通过普鲁士蓝染色及MRI细胞扫描发现,hATF-SPIO能够特异性结合Hela细胞且使得其T2WI信号明显减低;而对照组Lovo细胞MRI信号值在探针孵育前后未见明显变化,普鲁士蓝染色也未见hATF-SPIO在Lovo细胞中的滞留。MRI动物扫描显示hATF-SPIO探针能够随着血流达到HT29移植瘤且使得HT29肿瘤在T2加权相上较Lovo移植瘤信号降低,在探针注入后24h,两者信号值有明显差异;随后肿瘤组织的普鲁士蓝染色观察显示hATF-SPIO探针在HT29移植瘤内存留。综上所述,通过实验证明hATF-SPIO不仅在体外能够靶向结合uPAR表达的肿瘤细胞,并能够在体内靶向显影uPAR中度表达的结肠肿瘤。

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 nanopartiele （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 pathologi- cal 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 he subsequently in- ternalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal inten- sity 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.