Department of Medical Imaging and Nuclear Medicine, 4th Affiliated Hospital, Harbin Medical University, Harbin, 150001, China.
Mol Imaging Biol. 2010 Oct;12(5):520-9. doi: 10.1007/s11307-009-0286-0. Epub 2009 Dec 3.
The aim of this study is to monitor endostatin gene expression and therapy using transferrin receptor (TfR) as reporter gene and transferrin conjugate of ultrasmall supramagnetic iron oxide nanoparticle (Tf-USPIO) as magnetic resonance (MR) reporter probe.
A retroviral plasmid (pLP-LNCX) encoding mouse endostatin and TfR was constructed, and packaged with a titer of 4 × 10(7)colony-forming units per millimeter. MDA-MB-231 breast tumors were established in BALB/c mice by subcutaneous injection of 2 × 10(6) MDA-MB-231 cells. Mice were intratumorally injected with recombinant retrovirus and imaged with MR using Tf-USPIO. Western blot, Prussian blue, and immunohistochemical staining were performed to validate the magnetic resonance imaging results. The antitumor effect of retro-endostatin (ES)-TfR was also evaluated by intratumoral injection of the viral vector.
The expression of both endostatin and TfR genes in MDA-MB-231 cells after retroviral transfection was confirmed by Western blot and flow cytometry. Tf-USPIO conjugate binds specifically to cells stably transfected with retro-ES-TfR. After intravenous injection of the Tf-USPIO conjugate, there was a more pronounced decrease in T2 relaxation time in tumors treated with retro-ES-TfR than in tumors treated with empty retrovirus retro-LNCX. The expression of ES gene significantly delayed the growth of MDA-MB-231 tumor and reduction of microvessel density and VEGF level as compared to those without viral transfection or transfected with empty retro-LNCX vector.
Endostatin therapeutic gene expression was visualized successfully using TfR reporter gene and Tf-USPIO MR reporter probe, which indicates that MR reporter gene imaging may be valuable in gene therapy to evaluate therapeutic gene expression and treatment efficacy.
本研究旨在通过转铁蛋白受体（TfR）作为报告基因和转铁蛋白结合的超顺磁性氧化铁纳米颗粒（Tf-USPIO）作为磁共振（MR）报告探针，监测内皮抑素基因表达和治疗。
构建了编码小鼠内皮抑素和 TfR 的逆转录病毒质粒（pLP-LNCX），并包装成 4×10(7)个每毫米的集落形成单位。通过皮下注射 2×10(6)个 MDA-MB-231 细胞在 BALB/c 小鼠中建立 MDA-MB-231 乳腺肿瘤。通过 Tf-USPIO 进行 MR 成像，向肿瘤内注射重组逆转录病毒。通过 Western blot、普鲁士蓝和免疫组织化学染色验证磁共振成像结果。还通过瘤内注射病毒载体评估 retro-endostatin（ES）-TfR 的抗肿瘤作用。
Western blot 和流式细胞术证实逆转录病毒转染 MDA-MB-231 细胞后，内皮抑素和 TfR 基因的表达。Tf-USPIO 缀合物特异性结合稳定转染 retro-ES-TfR 的细胞。静脉注射 Tf-USPIO 缀合物后，与用空逆转录病毒 retro-LNCX 处理的肿瘤相比，用 retro-ES-TfR 处理的肿瘤 T2 弛豫时间明显降低。与未进行病毒转染或转染空 retro-LNCX 载体的肿瘤相比，ES 基因的表达显著延迟了 MDA-MB-231 肿瘤的生长，并降低了微血管密度和 VEGF 水平。
成功地使用 TfR 报告基因和 Tf-USPIO MR 报告探针可视化内皮抑素治疗基因表达，表明 MR 报告基因成像可能在评估治疗基因表达和治疗效果的基因治疗中具有重要价值。
