Division of Nuclear Medicine, University Hospital Leuven, Leuven, Belgium.
Nucl Med Biol. 2012 Nov;39(8):1266-74. doi: 10.1016/j.nucmedbio.2012.06.014. Epub 2012 Sep 14.
Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers.
The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET.
High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ~1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET.
We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.
过去十年间，正电子发射断层扫描（PET）成像技术在基因表达的研究中已崭露头角，成为生物医学研究的有力工具。单纯疱疹病毒 1 型胸苷激酶（HSV1-TK）是广泛应用的原型基因表达报告基因（PRG），同时还有许多其他 PRG 也已得到验证。我们研究了水痘带状疱疹病毒胸苷激酶（VZV-tk）作为新的 PRG，使用放射性标记的双环核苷类似物（BCNAs）作为 PET 示踪剂。
通过慢病毒载体（LV）转导后，在表达 VZV-tk 的细胞中和对照细胞中评估了四种不同放射性标记的 BCNAs 的摄取和洗脱情况。通过高压液相色谱（HPLC）测定示踪剂的代谢情况。用 PET 对携带表达 VZV-TK 的异种移植瘤的小鼠进行成像。
在所测试的 4 种示踪剂中，有 3 种在表达 VZV-tk 的细胞中表现出高摄取。在孵育溶液中存在过量胸苷时，可阻断示踪剂的摄取。细胞内保留率不同，其中一种示踪剂具有可接受的~1 小时半衰期。细胞内示踪剂的含量与用于转导细胞的 LV 滴度相关。通过 HPLC 显示出 VZV-TK 依赖性转化为代谢物。在表达含有 HSV1-TK 部分的融合蛋白的细胞中未观察到特异性积聚。在异种移植瘤中表达 VZV-tk 导致体内摄取增加 60%，这可通过 PET 测量到。
我们已经验证了 VZV-tk 和放射性标记的 BCNAs 作为新的 PRG/PRP 系统的组合。需要进一步优化 PRPs 和 PRG，以增加信号。
