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

在第一年，我们证实了位于辐射敏化启动子下游的基因表达确实会被辐射放大。我们构建了含E_4/CMV嵌合放射增敏启动子（E_4：位于Egr-1基因的CArG元件的四重重复结构）的EGFP和HSV-tk载体。将E_4/EGFP基因转染U251-MG/U373-MG细胞，3Gy照射后，荧光显微镜下观察EGFP的表达，Western blotting检测EGFP的表达。结果，我们证实，与照射前阶段相比，在6 × 54小时后，EGFP的表达扩增了约1.3 × 1.4倍。将E4/HSV-tk和HSV-tk分别转染U251-MG/U373-MG胶质瘤细胞系，MTT法检测GCV照射后（1、3、5Gy）细胞增殖情况。结果表明：（1）放射增敏启动子也能被物理或化学刺激激活; 2)E_4/HSV-tk组仅出现辐射剂量依赖性的细胞凋亡，但无统计学意义，第二年我们用<9ns-2>辐射增敏作用强于E_4的E_4替换启动子，进行同样的实验。但与预期相反，在E_ /EGFP -处理组中，EGFP的表达并没有显著增加<9ns-2>。MTT法也未证实E_<9ns-2>/HSV-tk对胶质瘤细胞株的增殖有明显抑制作用。本研究拟以E_4/CMV为放射增敏启动子，将E_4/HSV-tk基因导入C6和内皮前体细胞系，进一步推进我们的研究计划。

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.