中文摘要：

目的为以单纯疱疹病毒Ⅰ型胸苷激酶基因（HSV1-tk）为报告基因的核素心脏显像寻找合适的载体并提供依据。方法以胶原酶单次消化法获得乳鼠原代心肌细胞，分别应用重组腺病毒Ad5-HSV1-tk（Ad5-tk）和脂质体lipofectamine^TM2000包裹的重组质粒pDC316-tk（pDC316-tk／lipoplex）将报告基因HSV1-tk转导入细胞，以空载体腺病毒作为对照；采用Iodogen固相氧化法对特异性报告探针氟-碘阿糖呋喃基脲嘧啶（FIAU）进行131^I标记，经Sep-Pak C18反相色谱层析柱纯化后，测定标记物的标记率和放化纯；比较各组细胞对131^I-FIAU的摄取情况，并应用半定量反转录-聚合酶链反应（RT—PCR）和免疫细胞化学方法，在mRNA和蛋白质水平检测不同载体介导HSV1-tk转导心肌细胞后HSV1-tk的表达。结果131^I-FIAU的标记率为（53．82±2．05）％，放化纯为（94．85±1．76）％，标记物在体外稳定存在；转导后5h，Ad5-tk感染组和pDC316-tk／lipoplex转染组对131^I-FIAU的摄取率分别为（12．55±0．37）％、（2．09±0．34）％，且各时间点摄取率前者均明显高于后者（t=12．978～38．253，P均〈0．01）；2种载体介导心肌细胞转导后24h，RT—PCR即可检测到HSV1-tk mRNA的表达，且Ad5-tk感染组的表达水平明显高于pDC316-tk／lipoplex转染组（3．11±0．14与1．60±0．05，t=17．663，P〈0．01）；免疫细胞化学检测中二者阳性率分别为（81．70±0．40）％、（22．06±0．32）％，差异有统计学意义（t=23．237，P〈0．01）。结论腺病毒载体介导HSV1-tk感染心肌细胞的效率和对标记探针的摄取率均明显高于脂质体介导组，其可做为活体报告基因心脏显像的载体，可得到更为清晰的显像图。

英文摘要：

Objective Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In the present study, the recombinant plasmid and adenoviral vector carrying reporter gene, herpes simplex virus type 1 thymidine kinase （HSV1-tk） , were constructed and transducted into neonatal cardiac myocytes, and a series of in vitro studies were carried out on the cells transferred to evaluate the uptake of radiolabeled reporter probe and to compare both vectors for cardiac reporter gene imaging. Methods Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1- tk, chosen as the reporter gene, was inserted into adenovirus vector （Ad5-tk） and plasmid （pDC316-tk） , thus it could be transferred into neonatal cardiac myocytes. Recombinant adenovirus containing enhanced green fluorescent protein （AdS-EGFP） was used as control. Recombinant plasmid was coated with lipofectamine^TM 2000 （pDC316-tk/lipoplex）. The specific reporter probe of HSV1-tk, 2＇-fluoro-2＇-deoxy-1-β- D-arabinofuranosyl-uracil （FAU） , was labeled with 131^I by solid phase oxidation with Iodogen. Product was purified on a reverse-phase Sep-Pak C18 column and the radiochemical purity was then assessed. The accu- mulation of it in the transferred cardiac myocytes was detected as uptake rate. Furthermore, mRNA expression of HSV1-tk was detected by semi-quantitative reverse transcription-polymerase chain reaction （RT- PCR） , while its protein expression was located by immunoeytochemistry. Results FAU could be labeled with 131^I and the labeling efficiency was （53.82 ± 2.05） %. The radiochemical purity was （94.85 ± 1.76）% after purification, and it kept stable in vitro for at least 24 h. Time-dependent increase of the accumulation of 131^I-FIAU was observed in both Ad5-tk group and pDC316-tk/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. However, it also indicated that gr