Lan Xiaoli, Yin Xiaohua, Wang Ruihua, Liu Ying, Zhang Yongxue
Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Nucl Med Biol. 2009 Feb;36(2):207-13. doi: 10.1016/j.nucmedbio.2008.10.016.
Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images.
Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyluracil) was labeled with (131)I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [(131)I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry.
FAU could be labeled with (131)I, and the labeling efficiency and radiochemical purity rates were 53.82+/-2.05% and 94.85+/-1.76%, respectively. Time-dependent increase of the accumulation of [(131)I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55+/-0.37% and 2.09+/-0.34%, respectively. Greater uptakes of [(131)I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene expression by polymerase chain reaction in adenovirus vector-infected cardiac myocytes was significantly higher than that in pDC316-tk/lipoplex-transducted ones (semiquantitative analysis, 3.11+/-0.14 versus 1.60+/-0.05, P<.01). Immunocytochemistry showed that the transferred cardiac myocytes successfully expressed the target protein, and the positive rates were 81.70+/-0.40% in Ad5-tk and 22.06+/-0.32% in liposome (P<.01).
Both adenovirus and liposome could transfer reporter gene into cardiac myocytes successfully, and the expressed exogenous protein could form functional enzymes efficiently. However, the adenovirus vector acted more efficiently than did liposome, with a higher uptake rate of the reporter probe. Thus, adenovirus is competent for gene transfer in cardiac reporter gene imaging.
报告基因成像技术是一种用于心脏基因治疗无创监测的很有前景的方法。在本研究中，单纯疱疹病毒I型胸苷激酶（HSV1-tk）和2'-氟-2'-脱氧-1-β-D-阿拉伯呋喃糖基-5-碘尿嘧啶（FIAU）分别用作报告基因和探针。比较了腺病毒和脂质体两种载体转染HSV1-tk后新生大鼠心肌细胞对放射性标记FIAU的摄取情况。本研究旨在选择更好的载体，并为获得良好的核素图像提供理论依据。
通过单一胶原酶消化从大鼠心脏获取新生心肌细胞。构建了插入腺病毒载体（重组5型腺病毒，Ad5-tk）和用Lipofectamine 2000包被的质粒（pDC316-tk）（pDC316-tk/脂质体复合物），从而使HSV1-tk能够转入新生心肌细胞。用131I标记2'-氟-2'-脱氧-1-β-D-阿拉伯呋喃糖基尿嘧啶（FAU），并用反相Sep-Pak C-18柱纯化产物后进行评估。检测不同时间点（0.5、1、2、3、4和5小时）转染心肌细胞中[131I]FIAU的摄取率。此外，通过半定量逆转录聚合酶链反应和免疫细胞化学检测HSV1-tk的mRNA表达和蛋白表达。
FAU能够被131I标记，标记效率和放射化学纯度分别为53.82±2.05%和94.85±1.76%。在Ad5-tk组和pDC316/脂质体复合物组中均观察到[131I]FIAU的蓄积随时间增加，最高摄取率出现在5小时，峰值分别为12.55±0.37%和2.09±0.34%。在所有时间点，Ad5-tk感染细胞中[131I]FIAU的摄取均高于pDC316/脂质体复合物转染的细胞（t=12.978 - 38.253，P<0.01）。腺病毒载体感染的心肌细胞中通过聚合酶链反应检测到的外源基因表达显著高于pDC316-tk/脂质体复合物转导的细胞（半定量分析，3.11±0.14对1.60±0.05，P<0.01）。免疫细胞化学显示转染的心肌细胞成功表达了靶蛋白，Ad5-tk组的阳性率为81.70±0.40%，脂质体组为22.06±0.32%（P<0.01）。
腺病毒和脂质体均能成功将报告基因转入心肌细胞，且表达的外源蛋白能有效形成功能酶。然而，腺病毒载体的作用比脂质体更有效，报告探针的摄取率更高。因此，腺病毒适用于心脏报告基因成像中的基因转移。
