报告基因系统HSV1-sr39tk/FIAU监测治疗基因表达的体外研究

In vitro study on monitoring the expression of therapy gene via HSV1-sr39tk/FIAU report gene/probe system

构建HSV1-sr39tk为报告基因,以VEGF165为治疗基因的重组腺病毒载体Ad5-sr39tk-IRES-VEGF165(Ad5-SIV),将其以不同感染复数(MOI=0、10、25、50、75、100)感染大鼠骨髓间充质干细胞,用免疫激光共聚焦技术检测HSV1-sr39tk与VEGF165的表达。用酶联免疫吸附试验(ELISA)监测感染病毒后细胞培养上清中VEGF165蛋白分泌量;细胞摄取131I-FIAU实验监测HSV1-sr39tk报告基因的表达情况,以腺病毒包装增强型绿色荧光蛋白(Ad5-EGFP)为对照。结果显示:免疫共聚焦显微镜可观察到HSV1-sr39tk蛋白与VEGF165蛋白在细胞内成功表达;VEGF165蛋白分泌情况与MOI间呈线性正相关(P<0.05);感染Ad5-SIV后细胞对131I-FIAU摄取率增高与MOI间呈线性正相关(P<0.05)。不同时间摄取实验结果显示:30-150min间细胞摄取率迅速增高,之后增高减慢,进入平台期。MOI相关摄取结果与MOI相关ELLISA结果具有较好相关性(P<0.05)。Ad5-SIV经感染后可在大鼠骨髓间充质细胞中成功表达,并且治疗基因VEGF与报告基因HSV1-sr39tk的表达呈正相关,说明可通过报告基因HSV1-sr39tk监测治疗基因VEGF165的表达,为动物水平报告基因显像提供理论支持。

A recombinant adenovirus Ad5-sr39tk-IRES-VEGF165 （Ad5-SIV） was constructed in our lab, using mutant simplex herpes viral thymidine kinase reporter gene （HSVl-sr39tk） as a report gene, and human vascular endothelial growth factor 165 （VEGFI65） as a therapeutic gene. A replication-defective adenovirus carrying the enhanced green fluorescent protein gene （Ad5-EGFP） was used as a control. MSCs were infected with AdS-SIV at different multiplicity of infection （MOI = 0, 10, 25, 50, 75 and 100）. The expression of HSVl-sr39tk and VEGF165 was detected by immunofluorescence, and the secretion of VEGF was detected by enzyme-linked immunosorbent assay （ELISA）. The cellular uptake of 131I-FIAU was performed to detect the expression of HSVl-sr39tk. The immunofluorescence showed HSVl-sr39tk and VEGFI65 protein could express successfully at AdS-SlV-infected MSCs. The cellular uptake of 131I-FIAU increased with the virus titer（P〈0.05）. The time dependent uptake showed uptake rates increased rapidly from 30 min to 150 min, with a plateau after 150 min. ELISA results showed VEGFI65 protein secretion increased with the titer（P〈0.05）. The VEGFI65 protein secretion correlated highly with the uptake rate of 131I-FIAU（P〈0.05）. In conclusion, HSVl-sr39tk and VEGF165 could express successfully after MSCs were infected with Ad5-SIV. The expression of therapy gene correlated significantly with the reporter gene. This provides a theoretical basis for in vitro nuclear reporter gene imaging with this system.