H_2O_2诱导的PC12应激损伤中CR及SIRT1参与的调节效应  

Effects of CR on H_2O_2-Induced Stress Injury and Expression of SIRT1 in PC12 Cells

作　　者：李天题[1] 庄绪莹[1] 赵虎[1] 傅玉才[2] 李朝晖[1] 

机构地区：[1]中山大学中山医学院法医系,广州510080 [2]汕头大学医学院细胞衰老实验室,汕头515041

出　　处：《热带医学杂志》2010年第9期1051-1054,1072,F0003,共6页Journal of Tropical Medicine

摘　　要：目的研究H2O2诱导的PC12细胞(又称肾上腺嗜铬细胞瘤细胞)氧化应激损伤中,热量限制(caloric restriction,CR)参与的调控效应及SIRT1的表达变化。方法采用MTT法检测H2O2诱导的氧化应激损伤中CR对PC12细胞活力的影响;TUNEL染色法检测各实验组中PC12细胞的凋亡;免疫荧光检测PC12细胞中SIRT1的表达与定位;RT-PCR及Wester-blot检测各实验组中SIRT1、Caspase-3的表达。结果 60μmol/LH2O2作用6h后,H2O2组细胞活力可维持在70%以上,与正常对照组及CR组比较差异具有统计学意义;而120μmol/LH2O2作用下,H2O2组细胞活力显著下降。本实验选取60μmol/LH2O2浓度作为后续实验使用浓度。CR处理后PC12细胞的凋亡率较H2O2组显著降低。PC12细胞中SIRT1可于细胞浆和细胞核中表达,且主要于细胞浆中表达。H2O2作用6h后,与正常对照组相比SIRT1表达降低,Caspase-3表达上升;在CR+H2O2组中,与H2O2组比较,Caspase-3降低,SIRT1表达上升。结论 CR具有抗应激损伤及凋亡的效应,可上调PC12细胞中SIRT1的表达,在H2O2诱导的PC12细胞应激性损伤中CR-SIRT1的调控具有保护效应。Objective To study the regulatory effects of CR and the expression of SIRT1 in H2O2-induced stress injury.Methods MTT was used to detect survival rate of PC12 cells after pre-treating with H2O2; TUNEL-positive cells were detected using In Situ Cell Apoptosis Detection kit; Immunofluorescence staining method was used to detect the expression and localization of SIRT1; RT-PCR and Western-blot methods were used to detect the expression of SIRT1 and Caspase-3.Results After pretreating the cells with 60 μmol/L H2O2 for 6 h, the viability of PC12 cells in the H2O2 group was above 70%, and the result was significantly different from the CR＋H2O2 group.After treating the cells with 120 μmol/L H2O2 for 6 h, the viability of PC12 cells in the H2O2 group decreased remarkably.So 60 μmol/L H2O2 was selected for the subsequent studies.After treating with H2O2, TUNEL staining method showed that the rate of apoptosis in the CR ＋H2O2 group was decreased significantly when compared with the H2O2 group.Immunofluorescence staining results revealld that SIRT1 distributed more in the cytoplasm than in thenucleus; After treating with H2O2, the expression of SIRT1 was decreased and the expression of caspase-3 was increased.But in the CR＋H2O2 group, the caspase-3 level was decreased and the expression of SIRT1 was increased.Conclusion CR has anti-oxidative and anti-apoptosis effects.CR can upregulate the expression of SIRT1 and the effects of CR-SIRT1 can prevent PC12 cells from oxidative injury induced by H2O2.

关 键 词：热量限制 SIRT1 PC12 H2O2 

分 类 号：R592[医药卫生—老年医学]