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目的探讨硫氧还蛋白-1(sulfiredoxin-1,Srxn-1)对H2O2诱导的PC12细胞超氧化损伤的保护作用及其可能机制。方法在Lipofectamine 2000的介导下,分别将pLVT574、pLVT575和pLVT576干扰质粒转染PC12细胞,同时设空白对照组(正常培养细胞)及阴性对照组(转染pLVT4质粒),普通显微镜和倒置荧光显微镜下观察转染效率,并采用RT-PCR和Western blot法分别检测转染细胞中Srxn-1基因mRNA转录和蛋白表达的水平。采用不同终浓度(50、100、150、200、250、300μmol/L)的H2O2诱导PC12细胞,建立超氧化细胞损伤模型,MTT法检测Srxn-1对细胞存活率的影响,并采用超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)试剂盒检测细胞SOD活性及MDA含量。结果镜下观察可见,转染后细胞死亡数较多,胞体缩小;有绿色荧光,转染效率达50%。si-Srxn-1-575组细胞中Srxn-1基因mRNA转录及蛋白表达水平较空白对照组明显下降(P<0.05)。终浓度为200μmol/L的H2O2处理12 h后,PC12细胞的存活率为66%;H2O2+si-Srxn-1-575组的细胞存活率(49.3%)明显低于空白对照组(65.2%)(P<0.05)。H2O2+si-Srxn-1-575组细胞中SOD活性[(10.319±1.362)U/mg pro]明显低于H2O2+空白对照组[(15.7±1.138)U/mg pro],MDA含量[(5.507±0.297)nmol/mg pro]明显高于H2O2+空白对照组[(3.41±0.281)nmol/mg pro](P均<0.05)。结论 Srxn-1对H2O2损伤的PC12细胞具有保护作用,其机制可能与其发挥内源性抗氧化作用有关。
Objective To investigate the protective effect of sulfoxidoxin-1 (Srxn-1) on H2O2-induced superoxidative injury in PC12 cells and its possible mechanism. Methods PC12 cells were transfected with pLVT574, pLVT575 and pLVT576 plasmids respectively under the control of Lipofectamine 2000. At the same time, the blank control group (normal cultured cells) and the negative control group (pLVT4 plasmid transfected) were transfected with normal and inverted fluorescence microscopy The transfection efficiency was observed. The transcription and protein expression of Srxn-1 gene in transfected cells were detected by RT-PCR and Western blot respectively. The effect of Srxn-1 on cell viability was assayed by MTT assay, and the effect of superoxide dismutase (SOD) on the proliferation of PC12 cells was induced by different concentrations of H2O2 (50,100,150,200,250 and 300μmol / L) SOD activity and MDA content of superoxide dismutase (SOD) and malondialdehyde (MDA) Results The results of microscopic observation showed that the number of cell death after transfection was larger and the cell bodies were reduced; with green fluorescence, the transfection efficiency reached 50%. The mRNA and protein expression levels of Srxn-1 in si-Srxn-1-575 cells were significantly decreased compared with the blank control group (P <0.05). The survival rate of PC12 cells was 66% after treated with H2O2 at the concentration of 200μmol / L for 12 h. The cell viability (49.3%) in H2O2 + si-Srxn-1-575 group was significantly lower than that of the blank control group (65.2% (P <0.05). The activity of SOD in H 2 O 2 + si-Srxn-1-575 group was significantly lower than that in H 2 O 2 + blank control group [(15.7 ± 1.138) U / mg pro] [(10.319 ± 1.362) U / mg pro] 0.297) nmol / mg pro] was significantly higher than that of H2O2 + blank control group [(3.41 ± 0.281) nmol / mg pro] (all P <0.05). Conclusion Srxn-1 has a protective effect on PC12 cells injured by H2O2, and its mechanism may be related to its role in endogenous antioxidation.