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一种报告基因/探针系统 hERL/(18)F-FES 的体外和体内评估。

An in vitro and in vivo evaluation of a reporter gene/probe system hERL/(18)F-FES.

机构信息

Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

出版信息

PLoS One. 2013 Apr 12;8(4):e61911. doi: 10.1371/journal.pone.0061911. Print 2013.

DOI:10.1371/journal.pone.0061911
PMID:23593502
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3625158/
Abstract

PURPOSE: To evaluate the feasibility of a reporter gene/probe system, namely the human estrogen receptor ligand binding domain (hERL)/16α-[(18)F] fluoro-17β-estradiol ((18)F-FES), for monitoring gene and cell therapy. METHODS: The recombinant adenovirus vector Ad5-hERL-IRES-VEGF (Ad-EIV), carrying a reporter gene (hERL) and a therapeutic gene (vascular endothelial growth factor, VEGF165) through an internal ribosome entry site (IRES), was constructed. After transfection of Ad-EIV into bone marrow mesenchymal stem cells (Ad-EIV-MSCs), hERL and VEGF165 mRNA and protein expressions were identified using Real-Time qRT-PCR and immunofluorescence. The uptake of (18)F-FES was measured in both Ad-EIV-MSCs and nontransfected MSCs after different incubation time. Micro-PET/CT images were obtained at 1 day after injection of Ad-EIV-MSCs into the left foreleg of the rat. The right foreleg was injected with nontransfected MSCs, which served as self-control. RESULTS: After transfection with Ad-EIV, the mRNA and protein expression of hERL and VEGF165 were successfully detected in MSCs, and correlated well with each other (R(2) = 0.9840, P<0.05). This indicated the reporter gene could reflect the therapeutic gene indirectly. Ad-EIV-MSCs uptake of (18)F-FES increased with incubation time with a peak value of 9.13%±0.33% at 150 min, which was significantly higher than that of the control group. A far higher level of radioactivity could be seen in the left foreleg on the micro-PET/CT image than in the opposite foreleg. CONCLUSION: These preliminary in vitro and in vivo studies confirmed that hERL/(18)F-FES might be used as a novel reporter gene/probe system for monitoring gene and cell therapy. This imaging platform may have broad applications for basic research and clinical studies.

摘要

目的：评估报告基因/探针系统，即人雌激素受体配体结合域（hERL）/16α-[(18)F] 氟-17β-雌二醇（(18)F-FES），用于监测基因和细胞治疗的可行性。

方法：构建携带报告基因（hERL）和治疗基因（血管内皮生长因子，VEGF165）的重组腺病毒载体 Ad5-hERL-IRES-VEGF（Ad-EIV），通过内部核糖体进入位点（IRES）。将 Ad-EIV 转染入骨髓间充质干细胞（Ad-EIV-MSCs）后，采用 Real-Time qRT-PCR 和免疫荧光法鉴定 hERL 和 VEGF165 mRNA 和蛋白的表达。在不同孵育时间后，测量 Ad-EIV-MSCs 和未转染的 MSCs 对 (18)F-FES 的摄取。将 Ad-EIV-MSCs 注射到大鼠左前肢后 1 天获得微 PET/CT 图像。右前肢注射未转染的 MSCs，作为自身对照。

结果：Ad-EIV 转染后，成功检测到 MSCs 中 hERL 和 VEGF165 的 mRNA 和蛋白表达，且两者呈显著相关（R²=0.9840，P<0.05）。这表明报告基因可以间接反映治疗基因。Ad-EIV-MSCs 对 (18)F-FES 的摄取随孵育时间增加而增加，150 分钟时达到 9.13%±0.33%的峰值，明显高于对照组。微 PET/CT 图像上左前肢的放射性水平明显高于对侧前肢。

结论：这些初步的体外和体内研究证实，hERL/(18)F-FES 可作为监测基因和细胞治疗的新型报告基因/探针系统。这种成像平台可能在基础研究和临床研究中具有广泛的应用前景。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/57237a6d6cae/pone.0061911.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/935848c68d83/pone.0061911.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/bed01532c36e/pone.0061911.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/10dde5d47771/pone.0061911.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/aee27a360c6b/pone.0061911.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/57237a6d6cae/pone.0061911.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/935848c68d83/pone.0061911.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/bed01532c36e/pone.0061911.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/10dde5d47771/pone.0061911.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/aee27a360c6b/pone.0061911.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a69e/3625158/57237a6d6cae/pone.0061911.g005.jpg

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J Huazhong Univ Sci Technolog Med Sci. 2011-10

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