Moroz Maxim A, Serganova Inna, Zanzonico Pat, Ageyeva Ludmila, Beresten Tatiana, Dyomina Ekaterina, Burnazi Eva, Finn Ronald D, Doubrovin Michael, Blasberg Ronald G
Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
J Nucl Med. 2007 May;48(5):827-36. doi: 10.2967/jnumed.106.037812.
The norepinephrine transporter (NET) has recently been suggested as a useful reporter gene. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked hNET-green fluorescent protein (GFP) hybrid reporter gene for both nuclear and optical imaging.
A retroviral vector pQCXhNET-IRES-GFP was constructed and used to generate several reporter cell lines and xenografts. Transduced cells were sorted by fluorescence-activated cell sorting based on GFP expression and used for both in vitro and in vivo imaging studies.
The transduced reporter cells accumulated (123)I- or (124)I-labeled metaiodobenzylguanidine (MIBG) to high levels compared with the wild-type parent cell lines. Differences in MIBG accumulation between cell lines were primarily due to differences in influx (K(1)) rather than efflux (k(2)). The estimated MIBG distribution volumes (V(d)) for transduced Jurkat, C6, and COS-7 cells were 572 +/- 13, 754 +/- 25, and 1,556 +/- 38 mL/g, respectively. A correlation between radiotracer accumulation (K(1)) and GFP fluorescence intensity was also demonstrated. Sequential imaging studies of mice bearing pQCXhNET-IRES-GFP transduced and wild-type C6 xenografts demonstrated several advantages of (124)I-MIBG small-animal PET compared with (123)I-MIBG gamma-camera/SPECT. This was primarily due to the longer half-life of (124)I and to the retention and slow clearance (half-time, 63 +/- 6 h) of MIBG from transduced xenografts compared with that from wild-type xenografts (half-time, 12 +/- 1 h) and other organs (half-time, 2.6-21 h). Very high radioactivity ratios were observed at later imaging times; at 73 h after (124)I-MIBG injection, the C6/hNET-IRES-GFP xenograft-to-muscle ratio was 293 +/- 48 whereas the C6 xenograft-to-muscle ratio was 0.71 +/- 0.19.
These studies demonstrate the potential for a wider application of hNET reporter imaging and the future translation to patient studies using radiopharmaceuticals that are currently available for both SPECT and PET.
去甲肾上腺素转运体（NET）最近被提议作为一种有用的报告基因。我们通过构建一个用于核成像和光学成像的内部核糖体进入位点（IRES）连接的人NET-绿色荧光蛋白（GFP）杂交报告基因，扩展了这一研究。
构建逆转录病毒载体pQCXhNET-IRES-GFP，并用于生成多个报告细胞系和异种移植模型。基于GFP表达，通过荧光激活细胞分选对转导细胞进行分选，并用于体外和体内成像研究。
与野生型亲本细胞系相比，转导的报告细胞积累了高水平的（123）I或（124）I标记的间碘苄胍（MIBG）。细胞系之间MIBG积累的差异主要归因于流入（K（1））的差异，而非流出（k（2））的差异。转导的Jurkat细胞、C6细胞和COS-7细胞的估计MIBG分布容积（V（d））分别为572±13、754±25和1556±38 mL/g。还证实了放射性示踪剂积累（K（1））与GFP荧光强度之间的相关性。对携带pQCXhNET-IRES-GFP转导的异种移植模型和野生型C6异种移植模型的小鼠进行的连续成像研究表明，与（123）I-MIBGγ相机/SPECT相比，（124）I-MIBG小动物PET具有几个优势。这主要归因于（124）I的半衰期更长，以及与野生型异种移植模型（半衰期为12±1小时）和其他器官（半衰期为2.6 - 21小时）相比，MIBG从转导的异种移植模型中的保留和清除缓慢（半衰期为63±6小时）。在后期成像时间观察到非常高的放射性比值；在注射（124）I-MIBG后73小时，C6/hNET-IRES-GFP异种移植模型与肌肉的比值为293±48，而C6异种移植模型与肌肉的比值为0.71±0.19。
这些研究证明了hNET报告基因成像更广泛应用的潜力，以及未来使用目前可用于SPECT和PET的放射性药物转化为患者研究的可能性。
