新型核素报告基因系统hERL／核素标记雌二醇的实验研究  

Primary study of a novel reporter gene/probe system human ER ligand binding domain/radiolabledestradiol

作　　者：兰晓莉[1] 覃春霞[1] 田月丽[1] 夏晓天[1] 何勇[1] 袁辉[1] 张永学[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院核医学科、分子影像湖北省重点实验室、生物靶向治疗教育部重点实验室,武汉430022

出　　处：《中华核医学与分子影像杂志》2012年第1期10-15,共6页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：国家自然科学基金（30830041,30970853）

摘　　要：目的在体外细胞摄取实验及在体显像基础上，评价新型核素报告基因系统人ER配体结合域（hERL）／核素标记雌二醇（E2）应用的可行性，为其用于基因治疗的监测提供依据。方法以内部核糖体进入位点序列（IRES）方式构建携带hERL和治疗VEGF165的重组质粒pDC316-hERL-IRES．VEGF165（简写为EIV），将其用腺病毒包装构建重组腺病毒复合体（Ad-EIV）。采用悬浮贴壁法从sD大鼠股骨和胫骨原代提取骨髓MSCs并培养。将Ad-EIV和脂质体2000包裹重组质粒（Lipo-EIV）分别转染MSCs，利用RT-PCR和Westernblot，对hERL和VEGF165mRNA和蛋白质水平的表达分别进行检测。测定Ad．EIV组、Lipo．EIV组和未转染组MSCs对125I-E在不同时间（1、3,6、9、12和24h）的摄取率。将转染Ad-EIV的MSCs注射至大鼠左前肢、未转染MSCs注射至右前肢后1d，注射16a-^18TMF-17a-E2（^18F-FES）进行micro PET／CT活体显像。2组间均数比较采用t检验，相关性研究采用Pearson相关分析。结果Ad—EIV转染MSCs后RT—PCR检测示hERL和VEGFl65mRNA的表达随着感染滴度的增加而增加，且呈正相关（r2分别为0．953和0．966，P均〈0．05）；感染复数（MOI）=25、50、75和100时，其mRNA表达高于Lipo-EIV组。Westernblot检测蛋白质水平的表达也得到同样的结果。Ad-EIV组和Lipo-EIV组MSCs对^125I-E2的摄取率均随时间延长逐渐增高，24h最高，分别达到（10．94±0．30）％和（8．934-0．18）％；各个时间点2种载体转染组均明显高于未转染对照组（3．54％-5．52％；t值分别为15．489-26．560、10．523-24．204，P均〈0．05），而Ad-EIV组摄取率高于Lipo-EIV组（t=g．132—16．168，P均〈0．05）。MicroPET／CT大鼠活体显像显示注射Ad-EIV转染的MSCs之左前肢放射性浓聚明显高于对侧。结论报告基因hERL可以通过腺病毒转染、脂质体包裹等多种形式转染细胞并成功表达，应用核素标记E2可以对其�Objective To evaluate the feasibility of a new reporter gene/probe system, namely hu- man ER ligand binding domain （hERL）/radionuclide labeled estradiol, and to provide basis for its monito- ring gene and cell therapy from in vitro cellular uptake study and in vivo imaging experiment. Methods Recombinant plasmid pDC316- hERL -internal ribosome entry site-VEGF165 （pDC316-hERL-IRES-VEGF16s,or EIV） and recombinant Ad-EIV were constructed, which carried a reporter gene （hERL） and a therapeu- tic gene （VEGF165） through IRES. Adenovirus was used as a vector. MSCs were obtained from tibias and femurs of rat, and cultured normally. Ad-EIV and EIV coated with lipofectamine 2000 （Lipo-EIV） were transfected into MSCs. RT-PCR and Western blot were performed to detect the expression of hERL and VEGF165 from mRNA and protein level. The cellular uptake values of 125I labeled estradiol （^125-E2 ） were measured in Ad-EIV, Lipo-EIV and non-transfected MSCs at different incubation time （ 1,3,6, 9, 12 and24 h）. Ad-EIV transfected MSCs were injected into the left upper limb of rats, and non-transfected MSCs into the right upper limb as self-control. Micro PET/CT images were obtained after 1 d of transfection. T- test and Pearson linear correlation analysis were used to analyze the data. Results After transfected with Ad-EIV, mRNA and protein expressions of hERL and VEGF16s in MSCs were increased with adenovirus muhiplicity of infection （ MOI）, and positive correlation could be seen （ r^2 = 0. 953 and 0.966, both P 〈 0. 05）. The expressions of mRNA and protein in Ad-EIV group were higher than those of Lipo-EIV trans- fected MSCs. Time-dependent accumulation of 125 I-E2 was observed in the Ad-EIV group and Lipo-EIVgroup, and the highest uptake rates occurred at 24 h, with peak values of （ 10.94 ± 0. 30） % and （8.93± 0.18 ）% , respectively. Higher cellular uptakes could be seen at all time points in the Ad-EIV group than those of the Lipo-EIV group （t =4. 132- 16. 168, all P 〈0.05）.

关 键 词：基因 报告 雌激素受体配体结合域 放射性核素显像 雌二醇 同位素标记 大鼠 

分 类 号：R817.4[医药卫生—影像医学与核医学]