放射敏感性启动子调控CD基因/5-FC自杀系统慢病毒载体的构建及125I诱导表达的研究

摘要

合成了含8个CArG元件的放射敏感性启动子E8,将其连接于胞嘧啶脱氨酶(Cytosine Deaminase,CD)基因及GFP报告基因上游,构建了重组慢病毒载体pGC-FU- E8 codA- GFP.与慢病毒包装系统共转染293T细胞包装重组慢病毒颗粒E8 -coda- GFP LV,研究重组慢病毒感染EJ细胞在不同剂量125I的电离辐射下绿色荧光表达情况及其将5 -FC转化为5- FU的能力.结果显示:构建的含有E8启动子及CD基因的重组慢病毒载体,包装的重组慢病毒滴度为2×108 TU/mL；经125I照射的重组慢病毒感染EJ细胞均可观察到绿色荧光,其细胞上清液均可检测到5- FC转化成的5- FU紫外峰,其中55.5 kBq及74.0 kBq 125I照射的细胞组绿色荧光明显,148 kBq125I照射的细胞组,其5 -FU紫外峰最为明显.以上结果表明:本工作所构建的放射敏感性启动子调控CD 基因/5 -FC自杀系统重组慢病毒载体具有电离辐射调控作用,可在125I的电离辐射作用下诱导下游基因表达,为放射性核素125I联合CD基因/5 -FC自杀系统对肿瘤细胞的治疗作用研究提供了实验基础.%To package recombinant lentivirus, recombinant lentiviral vector carrying CD gene and GPP reporter gene and using synthetic radiation-responsive promoter which contains eight CArG elemenls(E8) as their upstream regulation sequence was constructed and transfected into 2.93T cells with pHelper 1.0 vector and pHelper 2. 0 vector. After EJ cells were infected by recombinant lentivirus and exposed to different doses of 125I, the expression of GFP gene was observed and the ability to converting 5-FC to 5-FU was assayed. The recombinant lentiviral vector was established successfully and the titer of recombinant lenti-virus was 2× 108 TU/mL. Exposure of lentivirus-infected EJ cells to 125'I, green fluorescence can be observed and 5-FU which was converted from 5-FC in cell supernatant can be detected. The green fluorescence was obviously observed at the dose of 55. 5 kBq and 74. 0 kBq, and the ultraviolet peak of 5-FU was the sharpest at the dose of 148 kBq. The results indicated that.synthetic promoter can induce the expression of downstream CD gene and reporter gene after exposure of l25I and provided a valuable contribution to the continued experiments on radionuclide125I therapy combination with CD gene/5-FC suicide gene therapy of tumor.