大分割剂量照射内皮细胞持续表达细胞因子模型的建立

Establishment of Endothelial Cell Model Which Continues to Express Cytokines Induced by Large Fractionated Radiation

目的:通过大分割剂量的X线照射人脐静脉血内皮细胞ECV-304,构建放射反应相关因子肿瘤坏死因子α(TNF-α)、转化生长因子β1(TGF-β1)持续表达模型,为探讨晚期放射反应中的分子靶点及干预策略提供基础。方法:大分割剂量的X线[5Gy/(次·周),连续6周,总计30Gy]照射指数生长期的ECV-304,在第一次照射后1,3,24,72h和后续每次照射后第5天及末次照射后第5,6,7,8周提取mRNA,采用qRT-PCR检测TNF-α、TGF-β1 mRNA水平的变化。于第一次照射后第1,3,24小时和后续每次照射后第5天,及末次照射后第4,6,8周收集细胞上清液,采用ELISA法测定其蛋白水平的表达情况。结果:正常ECV-304低表达TNF-α和TGF-β1。ECV-304细胞受到连续多次5Gy剂量照射后,于不同时间点,TNF-α和TGF-β1mRNA的表达均呈现先升高后降低的趋势,但始终高于正常水平。其中,TNF-αmRNA于末次照射后的第8周,表达量最高,为未照射时的(8.05±1.28)倍(P<0.05);而TGF-β1mRNA于末次照射后第5周升至高峰[为对照组(7.3±0.39)倍,P<0.05]。ECV-304细胞于单次照射后24h,TNF-α、TGF-β1蛋白表达量即开始升高,和mRNA趋势一致,TNF-α蛋白至第6次分割剂量照射后第5天表达量最高[(479.10±21.94)pg/ml(P<0.01)]。而TGF-β1蛋白于末次照射后6周升至高峰,蛋白含量为(2 335.93±126.18)pg/ml(P<0.01)。结论:TNF-α,TGF-β1在大分割剂量照射后,在mRNA和蛋白水平均明显升高,照射后8周仍持续升高,结果表明放射反应相关因子TNF-α、TGF-β1持续表达细胞模型构建成功。

Objective：To explore the new molecular targets to prevent and cure the late radiation inju-ries by establishing a continuous expression model of tumor necrosis factor-α（TNF-α）and transform growth factor-β1（TGF-β1）in ECV-304 cells induced by successive large fractionated irradiation.Methods：Total dose of 30Gy（5Gy per fraction per week）was delivered to ECV-304 cells,and the control group was sham-irradiated.At different time points between 1hr and 8weeks,the levels of TNF-α/TGF-β1mRNA and protein were measured by real-time RT-PCR and ELISA assay,respectively.Results：The sham-irradiated ECV-304 cells exhibited TNF-αand TGF-β1expression at a low level,whereas in ECV-304 cells exposed to successive large fractionated irradiation,the levels of TNF-αand TGF-β1mRNA increased gradually in the earlier period and declined in the later period,but were still significantly higher than those of the sham-irradiated cells at different time points,and TNF-α,TGF-β1mRNA reached the peak at 8th week with（8.05±1.28）folds（P〈0.05）and at 5th week with（7.3±0.39）folds（P〈0.05）as compared with the shamirradiated cells.In addition,the expression of TNF-αand TGF-β1protein began to ascend since the 24 th hour after the first radiation,the levels of TNF-αand TGF-β1protein rose to the peak at the 5th day after last irradiation with（479.10±21.94）pg/ml（P〈0.01）,and at the 6th week with（2 335.93±126.18）pg/ml（P〈0.01）,respectively.Conclusion：These results demonstrate the persistent expression of TNF-αand TGF-β1were induced by large fractionated radiation at different time points,even after 8weeks,indicating the cell-model which continues to express TNF-αand TGF-β1cytokines have been established successfully.