报告基因在大鼠心肌中表达的放射性自显影  

Autoradiography reporter gene （Ad5-tk carrying HSV1-tk gene ）expression imaging in rat myocardium

作　　者：刘影[1] 兰晓莉[1] 吴涛[1] 尹小花[1] 张永学[1] 

机构地区：[1]华中科技大学协和医院核医学科湖北省分子影像重点实验室,武汉430022

出　　处：《中华心血管病杂志》2009年第10期925-930,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金（30400176,30571816）

摘　　要：目的以单纯疱疹病毒1-胸苷激酶（HSV1-tk）为报告基因，以^131I标记的氟．碘阿糖基脲嘧啶（FIAU）为报告探针，对转染HSV1-tk基因的大鼠心肌组织行放射性自显影（ARG）研究，以探讨该报告系统用于心脏报告基因显像的可行性，并为显像的最佳病毒滴度和显像时间提供理论基础。方法制作腺病毒（Ad）为载体携带HSV1-tk基因的重组体Ad5-tk及空载病毒（Ad5-null），SD大鼠心肌内注射Ad5-tk及Ad5-null。根据目的不同进行分组如下：（1）转染报告基因后时间对显像的影响：心肌内转染1×10^8 pfu Ad5-tk后第1、2、3、5、7天注射^131I-FIAU；（2）转染不同病毒滴度对显像的影响：心肌内注射不同滴度的Ad5-tk（5×10^8，1×10^8，5×10^7，1×10^7pfu），2d后注射^131I-FIAU。对照组均采用注射同体积的Ad5-null。注射^131I-FIAU 24h后断颈法处死大鼠，取出心脏，测量γ计数值，行ARG研究，逆转录聚合酶链反应（RT—PCR）从mRNA水平证实，并对ARG图像和RT-PCR图像行半定量分析。结果ARG及RT-PCR图像上均可见局部报告基因的表达随感染滴度的增加而增高，随感染时间的延长而降低。半定量分析证实在滴度和时间上RT-PCR及γ计数与放射性自显影呈相关关系：不同滴度Ad5-tk转染大鼠心肌细胞，RT—PCR与ARG相关系数r^2=0．963，P〈0．05，γ计数与ARG相关系数r^2=0．996，P〈0．01；不同时间Ad5-tk转染大鼠心肌细胞，RT—PCR与ARG相关系数r^2=0．950，P〈0．05，γ计数与ARG相关系数r^2=0．980，P〈0．01。结论应用ARG方法证实HSV1-tk为报告基因，FIAU为报告探针的系统可以用于心肌报告基因显像，显像最佳的转染滴度为1×10^8pfu，最佳的显像时间为转染后24～48h，为在体进行报告基因心脏显像提供了理论基础。Objective 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 ^131I-2′-fluoro-2′-deoxy-1-β-D- arabinofuranosyl-5-iodouracil （^131I-FIAU） and autoradiography （ARG）. Methods The recombinant Ad5-tk carrying HSV1-tk gene and adenovirus （Ad5-null） as vector were constructed and intramyoeardially 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 ^131I-FIAU at day 1, 2, 3, 5 and 7 after transfection of 1 × 10^8pfu Ad5-tk; ② Influence of various titers on the imaging： rats underwent intramyoeardial injection with various titers of Ad5- tk（5× 10^8 , 1 × 10^8 ,5 × 10^7 , 1 × 10^7pfu）. After ：2 days, rats were injected with ^131I-FIAU in tail vein. Equal volume Ad-nulls was intramyocardially injected to control rats. Rats were killed 24 h after injection of ^131I-FIAU and the hearts were rapidly dissected for gamma counts measurement. The total myocardial ^131I-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. Results 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

关 键 词：心脏 基因 报告 放射自显影术 

分 类 号：R686[医药卫生—骨科学]