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

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2×10⁸TU/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 kBq ¹²⁵I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。

Construction and 125I Induced-expression in EJ Cells of Lentiviral Vector Carrying Radiation-responsive Promoter and CD Gene

To package recombinant lentivirus, recombinant lentiviral vector carrying CD gene and GFP reporter gene and using synthetic radiation-responsive promoter which con- tains eight CArG elements（E8） as their upstream regulation sequence was constructed and transfected into 293T 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 expres- sion 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 lentivirus was 2 ×10^8 TU/mL. Exposure of lentivirus-infected EJ cells to 125I, green fluores- cence 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 125I and provided a valuable contribution to the continued experiments on radionuclide 125I therapy combination with CD gene/5-FC suicide gene thera- py of tumor.