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

ZHANG Guopeng LAN Xiaoli CHEN Quan CAO Wei HE Yong ZHANG Yongxue

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

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- In vitro study on monitoring the expression of therapy gene via HSV1-sr39tk/FIAU report gene/probe system
- NUCLEAR TECHNIQUES Vol. 33, Issue 8, (2010)
- 作者机构：
  
  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
- 作者简介：
- 基金信息：
  
  国家自然科学基金(30571816,30772208,30830041)资助
- DOI：
  CLC： R346
- Published：01 August 2010
- 稿件说明：
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[1]张国鹏,兰晓莉,陈铨,曹卫,何勇,张永学.报告基因系统HSV1-sr39tk/FIAU监测治疗基因表达的体外研究[J].核技术,2010,33(08):592-597.

ZHANG Guopeng LAN Xiaoli CHEN Quan CAO Wei HE Yong ZHANG Yongxue. In vitro study on monitoring the expression of therapy gene via HSV1-sr39tk/FIAU report gene/probe system[J]. Nuclear techniques, 2010, 33(8): 592-597.
[1]张国鹏,兰晓莉,陈铨,曹卫,何勇,张永学.报告基因系统HSV1-sr39tk/FIAU监测治疗基因表达的体外研究[J].核技术,2010,33(08):592-597. DOI：

ZHANG Guopeng LAN Xiaoli CHEN Quan CAO Wei HE Yong ZHANG Yongxue. In vitro study on monitoring the expression of therapy gene via HSV1-sr39tk/FIAU report gene/probe system[J]. Nuclear techniques, 2010, 33(8): 592-597. DOI：

摘要

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。

Abstract

A recombinant adenovirus Ad5-sr39tk-IRES-VEGF165(Ad5-SIV) was constructed in our lab

using mutant simplex herpes viral thymidine kinase reporter gene(HSV1-sr39tk) as a report gene

and human vascular endothelial growth factor 165(VEGF165) 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 Ad5-SIV at different multiplicity of infection(MOI = 0

75 and 100).The expression of HSV1-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 HSV1-sr39tk.The immunofluorescence showed HSV1-sr39tk and VEGF165 protein could express successfully at Ad5-SIV-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 VEGF165 protein secretion increased with the titer(P<0.05).The VEGF165 protein secretion correlated highly with the uptake rate of 131I-FIAU(P<0.05).In conclusion

HSV1-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.

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