基于受体的报告基因的基因转移的多模态成像。

Multimodality imaging of gene transfer with a receptor-based reporter gene.

机构信息

Department of Radiation Oncology, School of Medicine, Washington University, St. Louis, Missouri 63108, USA.

出版信息

J Nucl Med. 2010 Sep;51(9):1456-63. doi: 10.2967/jnumed.109.063586. Epub 2010 Aug 18.

DOI:10.2967/jnumed.109.063586

PMID:20720053

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2981350/

Abstract

UNLABELLED

Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Noninvasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor-enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging.

METHODS

An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of (111)In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice.

RESULTS

The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of (111)In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2. Although fluorescence was observed, there were discrepancies between in vivo imaging and ex vivo imaging as well as between nuclear imaging and fluorescent imaging.

CONCLUSION

These studies showed that the SSTR2-EGFP fusion construct can be used for in vivo nuclear and optical imaging of gene transfer.

摘要

目的

构建并评估携带生长抑素受体 2（SSTR2）-增强型绿色荧光蛋白融合基因（SSTR2-EGFP）的腺病毒载体（AdSSTR2-EGFP），探讨其体内外转染及核素与荧光双模式成像的可行性。方法：构建携带 SSTR2-EGFP 的重组腺病毒载体（AdSSTR2-EGFP），采用体外细胞培养实验、动物模型体内实验，对其进行检测。采用 SCC-9 人鳞癌细胞，进行细胞结合实验、内吞实验和荧光分光光度计检测，评估 AdEGFP、AdSSTR2 和 AdSSTR2-EGFP 在体外的表达情况。通过小动物活体成像仪和单光子发射计算机断层扫描（SPECT），研究裸鼠 SCC-9 移植瘤模型瘤内注射 AdSSTR2-EGFP 或 AdSSTR2 后的体内分布和放射性核素示踪，比较其与放射性核素标记的生长抑素类似物（111）In-二乙三胺五乙酸-奥曲肽（111In-DTPA-Tyr3-octreotate）在体内的靶向性。裸鼠瘤内注射 AdSSTR2、AdSSTR2-EGFP 或 AdEGFP 后，行活体荧光成像，处死动物后提取组织标本，行离体荧光成像。结果：体外细胞培养实验结果显示，SSTR2-EGFP 与野生型 AdSSTR2 的受体结合及内吞能力相似，SSTR2-EGFP 感染细胞的荧光发射光谱与野生型 AdEGFP 感染细胞几乎完全一致。SPECT 及放射性核素分布实验显示，5 只裸鼠 SCC-9 移植瘤模型瘤内分别注射 AdSSTR2 或 AdSSTR2-EGFP 后，放射性核素 111In-DTPA-Tyr3-octreotate 在肿瘤内的摄取差异无统计学意义（P > 0.05），明显高于对照组（未转染的肿瘤）。裸鼠瘤内注射 AdSSTR2-EGFP 或 AdEGFP 后，在体内及离体状态下均可观察到荧光，瘤内注射 AdSSTR2 后则未见荧光。虽然可观察到荧光，但体内与离体、核素与荧光成像之间存在差异。结论：本研究构建的 SSTR2-EGFP 融合基因表达载体可用于基因转染的体内核素与光学成像。