^99Tc^m-亚锡葡庚糖酸钠结合GGC序列监测基因治疗的实验研究  

The experimental study of monitoring gene-based therapy with the binding of diglycylcysteine binding motifs and 99Tcm-glucoheptonate

作　　者：张国鹏[1] 兰晓莉[1] 何勇[1] 陈铨[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、湖北省分子影像重点实验室,武汉430022

出　　处：《中华核医学杂志》2011年第2期128-133,共6页Chinese Journal of Nuclear Medicine

基　　金：国家自然科学基金（30571816,30772208,30830041）

摘　　要：目的 构建以金属结合肽（双甘氨酰半胱氨酸,即GGC序列）为核素报告基因、人VEGF165为治疗基因的重组腺病毒载体,以99Tcm-GH为报告探针,探讨该报告系统监测治疗基因表达的可行性.方法 pcDNA3-VEGF165质粒线性化后,将金属结合肽GGC序列连于VEGF165基因C端,通过内部核糖体切入位点（IRES）连接增强型绿色荧光蛋白（EGFP）,构建重组腺病毒载体Ad5-VEGF165GGCmotif-IRES-EGFP（Ad5-VIE）,同时构建腺病毒包装EGFP（Ad5-EGFP）为对照.以不同感染复数（MOI=0,10,25,50,100）Ad5-VIE感染大鼠骨髓间充质干细胞（MSC）后,用99Tcm-GH为报告探针,研究感染细胞摄取动力学（30,60,90和120 min）情况,以检测GGC序列在MSC中的表达,并与实时定量PCR、Western-blot蛋白印迹、免疫组织化学等方法鉴定的VEGF165表达进行对比分析.通过荧光显微镜及实时定量PCR检测EGFP在细胞中的表达.采用SPSS 13.0软件进行统计学处理,组间比较运用独立样本成组t检验、q检验和直线（Pearson）相关分析.结果 以Ad5-VIE感染MSC后,不同MOI摄取实验结果示细胞对99Tcm-GH的摄取率随病毒MOI的增加而逐渐增加（r2=0.86,P＜0.05）,并且在MOI=100时达到（7.94±0.75）%;不同时间摄取实验示随99Tcm-GH孵育时间延长,细胞摄取率逐步增高,至120 min时达到（7.72±0.22）%.Ad5-VIE感染组与Ad5-EGFP感染组摄取率在各个不同时间点差异均有统计学意义（t=15.10～54.92,P均＜0.05）.在mRNA水平上,VEGF165及EGFP表达均随病毒MOI增加而增加（r2=0.99,P＜0.05）.不同MOI下细胞对99Tcm-GH的摄取与VEGF165蛋白表达呈较好的相关性（r2=0.90,P＜0.05）.免疫组织化学检查结果表明人VEGF165目的 基因在MSC中成功表达.荧光显微镜下可以观测到被感染细胞中的EGFP蛋白.结论 成功构建的重组腺病毒系统Ad5-VIE感染MSC对99Tcm-GH的摄取与VEGF165表达呈正相关.以GGC多肽为报告基因可以监测治疗基因VEGF165的表达,为Objective To evaluate the feasibility of monitoring the gene expression of VEGF165 via the diglycylcysteine （GGC） reporter gene system by reporter probe of 99Tcm-GH. Methods DNA fragments encoding GGC binding motifs were prepared by PCR and positioned at the C end of VEGF165 gene after the linearization of pcDNA3-VEGF165 plasmid. A replication-defective adenovirus vector Ad5-VEGF165GGC motif-internal ribosomal entry site（IRES） -enhanced green fluorescent protein （EGFP） （Ad5-VIE）was constructed, with a cytomegalovirus （CMV） early promoter driving the expression of VEGF165 gene,GGC motif and EGFP, under the aid of an IRFS. A replication-defective adenovirus carrying the Ad5-EGFP was used as the control. Mensenchymal stem cells （MSC） were infected with the recombinant adenovirus at a multiplicity of infection （MOI） from 0 to 100 infectious units （0,10,25,50,100）. The cellular uptake of 99Tcm-GH in infected MSC were then studied at 30, 60, 90 and 120 min. VEGF165 was detected by quantitative reverse transcriptase real-time PCR （RT-PCR）, Western-blot, and immunohistochemisty. EGFP was observed by RT-PCR and fluorescence microscopy. The correlation analysis was studied between the cellular uptake of 99Tcm-GH and the expression of VEGF165. SPSS 13.0 was applied for statistical analysis. Independent samples t-test, q-test and Pearson correlation coefficient were used. Results After infected with different viral titer of Ad-VIE, the cellular uptake of 99Tcm-GH increased with the increasing virus titer（r2 =0.86, P 〈0.05）, with the peak rate （7.94 ±0.75） % at MOI = 100. In time-dependent uptake study, the cellular uptake rates increased rapidly with the time extension, and the highest uptake occurred at 120 min with the peak uptake rate （7.72 ±0.22）%. The uptake rates of 99Tcm-GH in Ad5-VIE-infected cells were significantly higher than those of Ad5 -EGFP-transfected cells at all time points （t = 15.10- 54.92, all P 〈0.05）. The VEGF165 and EGFP mRNA levels increased w

关 键 词：甘氨酸 半胱氨酸 肽类 基因 报告 葡庚糖酸盐 锝 大鼠 

分 类 号：R450[医药卫生—治疗学]