Combination of radiation and suicide gene therapy with radio-sensitized promoter/Endotherial precursor cell in glioma

在第一年，我们验证了位于放射致敏启动子下游的基因表达实际上会被辐射放大。我们构建了含有E_4/CMV嵌合无线电致敏启动子（E_4：位于Egr-1基因上的CArG元件的四重复结构）的egfp和hsv -tk载体。将E_4/EGFP分别转入U251-MG/U373-MG人胶质瘤细胞系，经3Gy照射后，采用荧光显微镜光学观察和Western blot检测EGFP的表达。结果证实，与辐照前相比，6 ~ 54小时后EGFP的表达量增加了1.3 ~ 1.4倍。将E4/HSV-tk和HSV-tk分别转染到U251-MG/U373-MG人胶质瘤细胞系中，用MTT法观察GCV给药后1、3、5Gy辐照对细胞增殖的影响。结果表明：1)放射性致敏启动子也能被物理/化学刺激激活。2)辐射剂量依赖性凋亡仅在E_4/ hsv -tk给药组存在，但无统计学意义。第二年，我们用比E_4更强的E_<9ns-2>代替启动子，进行同样的试验。然而，与预期相反，EGFP的表达在E_<9ns-2>/EGFP -管理组中没有显著扩增。在MTT实验中，我们也没有证实辐照对E_<9ns-2>/ hsv -tk-给药胶质瘤细胞株的细胞增殖有显著的抑制作用。我们将利用E_4/CMV作为放射致敏启动子，将E_4/HSV-tk转移到C6和内皮前体细胞中，推进我们之前的计划。

In the first year we verified that the gene expression located at the downstream of radio-sensitized promoter would actually amplify by radiation. We constructed the EGFP&HSV-tk vectors containing E_4/CMV chimeric radio-sensitized promoter (E_4: four-repeated structure of CArG element located at Egr-1 gene). E_4/EGFP was transferred into U251-MG/U373-MG human glioma cell line respectively and the expression of EGFP was evaluated by optical observation under fluorescent microscopy and Western blotting after 3Gy irradiation. As a result, we verified that the expression of EGFP was amplified about 1.3〜1.4 times after 6〜54 hours compared to pre-irradiation stage. Then E4/HSV-tk and HSV-tk were transferred into U251-MG/U373-MG human glioma cell line respectively, and the degree of cell proliferation was evaluated by MTT assay after irradiation (1,3,5Gy) following GCV administration. The results were 1)the radio-sensitized promoter also could be activated by physical/chemical stimuli. 2)the radiation dose-dependent apoptosis was observed only in the E_4/HSV-tk-administrative group but not statistically significant.In the second year, we replaced the promoter with E_<9ns-2>, stronger than E_4 in radio-sensitization and did the same trial. Contrary to the expectation, however, the expression of EGFP was not significantly amplified in the E_<9ns-2>/EGFP -administrative groups. In the MTT assay, we also could not verified the significant inhibition of the cell proliferation among E_<9ns-2>/HSV-tk-administrative glioma cell lines after irradiation. We are going to promote our previously scheduled plan by using E_4/CMV as the radio-sensitized promoter, transferring E_4/HSV-tk into C6 and endothelial precursor cell line.