Liu Ying, Lan Xiao-li, Wu Tao, Yin Xiao-hua, Zhang Yong-xue
Department of Nuclear Medicine, Union Hospital, Huazhong University of Science and Technology, Hubei Key Laboratory of Molecular Imaging, Wuhan 430022, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2009 Oct;37(10):925-30.
Radionuclide imaging of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Herpes simplex virus 1-thymidine kinase (HSV1-tk) has been successfully applied to the tumor tissue.We explored the feasibility of the expression imaging of HSV1-tk reporter gene in rat myocardium by using SPECT reporter probe (131)I-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil ((131)I-FIAU) and autoradiography (ARG).
The recombinant Ad5-tk carrying HSV1-tk gene and adenovirus (Ad5-null) as vector were constructed and intramyocardially injected into SD rats. Experiment was grouped for different aims as follows: (1) Influence of time on the imaging after transfection reporter gene: rats were injected with (131)I-FIAU at day 1, 2, 3, 5 and 7 after transfection of 1 x 10(8)pfu Ad5-tk; (2) Influence of various titers on the imaging: rats underwent intramyocardial injection with various titers of Ad5-tk (5 x 10(8), 1 x 10(8), 5 x 10(7), 1 x 10(7)pfu). After 2 days, rats were injected with (131)I-FIAU in tail vein. Equal volume Ad-nulls was intramyocardially injected to control rats. Rats were killed 24 h after injection of (131)I-FIAU and the hearts were rapidly dissected for gamma counts measurement. The total myocardial (131)I-FIAU accumulation was quantified in percent of injected dose per gram myocardium (%ID/g). The myocardial reporter gene expression was semi-quantitatively determined by ARG and RT-PCR.
ARG and RT-PCR showed that the local expression of reporter gene increased in proportion with increasing titer and decreased in proportion with time post injection. The semi-quantitative assay showed there were significant correlations among %ID/g, RT-PCR and ARG: r(2) = 0.963, P < 0.05 for RT-PCR and ARG; r(2) = 0.996, P < 0.01 for %ID/g and ARG in rats received various reporter gene titers at identical time point post injection; r(2) = 0.950, P < 0.05 for RT-PCR and ARG; r(2) = 0.980, P < 0.01 for %ID/g and ARG for rats received identical reporter gene titer on various time points post injection.
The present study showed that cardiac reporter gene imaging with HSV1-tk as reporter gene and (131)I-FIAU as reporter probe was feasible in rats. The optimal Ad5-tk titer is 1 x 10(8) pfu and the optimal imaging time is 24 h to 48 h post gene transfer. HSV1-tk/FIAU may be used for the noninvasive monitoring of cardiac gene therapy.
报告基因表达的放射性核素成像有望用于基因治疗的无创监测。单纯疱疹病毒1 - 胸苷激酶（HSV1 - tk）已成功应用于肿瘤组织。我们通过使用单光子发射计算机断层扫描（SPECT）报告探针131I - 2'-氟 - 2'-脱氧 - 1 - β - D - 阿拉伯呋喃糖基 - 5 - 碘尿嘧啶（131I - FIAU）和放射自显影（ARG），探讨HSV1 - tk报告基因在大鼠心肌中表达成像的可行性。
构建携带HSV1 - tk基因的重组腺病毒Ad5 - tk及作为载体的腺病毒（Ad5 - null），经心肌内注射到SD大鼠体内。根据不同目的进行实验分组如下：（1）时间对转染报告基因后成像的影响：在转染1×10⁸ 空斑形成单位（pfu）Ad5 - tk后的第1、2、3、5和7天给大鼠注射131I - FIAU；（2）不同滴度对成像的影响：给大鼠经心肌内注射不同滴度的Ad5 - tk（5×10⁸、1×10⁸、5×10⁷、1×10⁷ pfu）。2天后，经尾静脉给大鼠注射131I - FIAU。给对照大鼠经心肌内注射等体积的Ad - null。在注射131I - FIAU后24小时处死大鼠，迅速取出心脏进行γ计数测量。以每克心肌注射剂量的百分比（%ID/g）对心肌中131I - FIAU的总蓄积量进行定量。通过ARG和逆转录 - 聚合酶链反应（RT - PCR）对心肌报告基因表达进行半定量测定。
ARG和RT - PCR显示，报告基因的局部表达随滴度增加而升高，随注射后时间延长而降低。半定量分析显示，%ID/g、RT - PCR和ARG之间存在显著相关性：在注射后相同时间点接受不同报告基因滴度的大鼠中，RT - PCR与ARG的r² = 0.963，P < 0.05；%ID/g与ARG的r² = 0.996，P < 0.01；在注射后不同时间点接受相同报告基因滴度的大鼠中，RT - PCR与ARG的r² = 0.950，P < 0.05；%ID/g与ARG的r² = 0.980，P < 0.01。
本研究表明，以HSV1 - tk作为报告基因、131I - FIAU作为报告探针的心脏报告基因成像在大鼠中是可行 的。最佳Ad5 - tk滴度为1×10⁸ pfu，最佳成像时间为基因转移后24小时至48小时。HSV1 - tk/FIAU可用于心脏基因治疗的无创监测。
