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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。
Objective To search for genes differentially expressed in human lung fibroblasts (MRC-5) induced by low-dose hydrogen peroxide (H2O2). Methods The cell viability was measured by MTT method to determine the dose-response relationship between H2O2 and MRC-5 toxicity. The cells were challenged with low dose, high dose and low dose, and then challenged with high dose to observe cell growth The adaptive response model of MRC-5 induced by low-dose H2O2 was obtained. Differentially expressed genes stimulated differently were then searched using FluoroDD-PCR. Results According to the dose-response relationship of MRC-5 toxicity induced by H2O2, low dose of 0.088,0.8,8.8,88μmol / L and high dose of 1100μmol / L were selected. Cells were treated with low dose for 24h and then stimulated with high dose for 1 hour. After the cells were pre-stimulated at 0.88 μmol / L for 24 h, there was a significant resistance effect on the subsequent stimulation of 1100 μmol / L. FluoroDD-PCR was performed on differently treated cell RNAs to find 60 differential bands. Fifteen of them were cloned and identified for homology. Among them, seven known genes and eight new genes were found. Conclusions H2O2 can induce the adaptive reaction of MRC-5 and find the differentially expressed genes by FluoroDD-PCR.