硫氧还蛋白还原酶2基因抗砷作用

Study on arsenic tolerance of thioredoxin reductase 2 gene in arsenic resistance ECV 304 cells

目的研究硫氧还蛋白还原酶2(thioredoxin reductase,TrxR2)基因在抗砷细胞(As-ECV304)中的抗砷作用。方法蛋白印迹法(Western Blot)检测As-ECV304和对照组细胞TrxR2蛋白水平,流式细胞仪检测细胞周期分布,化学合成TrxR2基因小干扰RNA(small interfering RNA,siRNA)转染As-ECV304细胞,Western Blot验证干扰效率,细胞急性毒性试验检测细胞抗砷性的改变。结果As-ECV304细胞中TrxR2蛋白水平较对照组细胞高,As-ECV304细胞G0/G1期细胞所占比例高于对照细胞。3个siRNA中有一个能特异性抑制TrxR2基因表达,干扰组细胞的生存率低于阴性对照组和未干扰组,3组细胞的半数抑制率IC50分别为9.13,15.88和18.52μmol/L。结论siRNA干扰TrxR2基因表达后能明显降低抗砷细胞的抗砷性,提示该基因在细胞抗砷机制中发挥了重要作用。

Objective To study the function of thioredoxin reductase 2 （TrxR2） gene in arsenic - resistance （ As - ECV304） ceils. Methods The protein level of TrxR2 in As - ECV300 and control ECV304 cells was detected by Western Blot and the cell cycles were detected by flowcytometry （FCM）. Three short interfering RNA （siRNA） of TrxR2 gene and negative control siRNA were made from chemical synthesis. As - ECV 300 cells transfected with 3 TrxR2 siRNA and control siRNA were exposed to NaAsO2 for 72 hours and the effect of RNA interference was studied by Western Blot. As - ECV 300 Cells transfected secondly with the TrxR2 siRNA,control siRNA and un-transfected were cultured for 24 hours and then exposed to NaAsO2 for 24 hours. Cell viability and arsenic tolerance were analyzed by MTT assay, Results Western Blot showed that the TrxR2 protein level was higher in As - ECV 304 cells than in control ECV 304 Cells. Cell cycle of As - ECV304 cell and control cell showed different percent in phase and the percent of G0/G1 phase was higher in As - ECV304 cells. One of three TrxR2 siRNA can specially interfered target gene expression. MTT assay showed that the cell viability was obviously decreased in cells transfected with TrxR2 siRNA than control siRNA. The 50% inhibiting concentration （IC50） of As - ECV 300 cells transfected with TrxR2 siRNA, control siRNA and without transfection exposed to NaAsO2 was 9.13 μmol/L, 15.8 μmol/L and 18.52 μmol/L, respectively. Conclusion The arsenic tolerance of As - ECV 300 cells transfected with TrxR2 siRNA significantly decreased, the TrxR2 gene may play an important role in arsenic tolerance of As - ECV 300 cells.