低剂量过氧化氢诱导机体适应性反应差异表达基因的研究

Research on differential display genes of adaptive response induced by hydrogen peroxide

目的寻找低剂量过氧化氢(H2O2)诱导人胚肺成纤维细胞(MRC-5)适应性反应差异表达的基因。方法用溴化四氮唑蓝(MTT)法检测细胞存活率得到H2O2对MRC-5毒性的剂量反应关系,分别用低剂量、高剂量和低剂量预刺激后用高剂量攻击细胞,观察细胞生长的变化,得到低剂量H2O2诱导MRC-5的适应性反应模型。然后用荧光差异显示聚合酶链反应(FluoroDD-PCR)寻找不同方式刺激的差异表达基因。结果根据H2O2诱导MRC-5毒性的剂量反应关系,选择0.088、0.88、8.8、88μmol/L为低剂量,1100μmol/L为高剂量,用低剂量处理细胞24h,然后用高剂量刺激1h,可以观察到在0.88μmol/L预刺激细胞24h后,对继而1100μmol/L的刺激有明显的抵抗效应。对不同方式处理的细胞RNA进行FluoroDD-PCR,找到60个差异条带。对其中的15个差异条带进行克隆鉴定同源性比较,发现其中7个已知基因,8个新基因。结论H2O2可以诱导MRC-5的适应性反应过程,用FluoroDD-PCR方法找到差异表达的基因。

ObjectiveTo search the differential expressed genes of low concentration of hydrogen peroxide (H_2O_2) inducing the human embryo lung fibroblast (MRC-5). MethodsWith the method of MTT, the dose-effect relation of H_2O_2 toxicity to MRC-5 was built by means of MTT. The cellular viability of MRC-5 was observed after being treated separately with H_2O_2 by low concentration, high concentration, low concentration pretreatment then high concentration attack. When adaptive response model was selected, fluorescence differential display polymerase chain reaction (Fluoro DD-PCR) was used to search differential expressed genes of the different treatment groups of H_2O_2. ResultsBased on the dose-effect relation of H_2O_2 to MRC-5, (0.088), 0.88, 8.8, 88 μmol/L were chosen as low concentration, 1 100 μmol/L as high concentration . MRC-5 was pretreated with low concentration for 24 hours, and then attacked with high concentration for 1 hour .Cellar viability was observed and adaptive response model was found and 60 differential expressed straps were acquired by fluoro DD-PCR. 15 differential straps were cloned and sequenced. 7 of them were known genes, while 8 of them were new ones. ConclusionH_2O_2 could induce adaptive response of MRC-5, differential straps could be identified to provide scientific base for deepening the research of the mechanism of low concentration H_2O_2 inducing adaptive response of MRC-5.