PC12细胞应激损伤中能量限制的效应及SIRT3的表达  

Effects of caloric restriction on the oxidative stress injury and the expression of SIRT3 in PC12 cell

作　　者：李天题[2] 李朝晖[1] 庄绪莹[1] 傅玉才[1] 

机构地区：[1]中山大学中山医学院法医系,广州510080 [2]汕头大学医学院第一附属医院病理科

出　　处：《中华医学杂志》2011年第5期350-354,共5页National Medical Journal of China

摘　　要：目的 研究H2O2诱导的PC12细胞氧化应激损伤中,能量限制（CR）及SIRT3参与的调控效应.方法 实验分4组：H2O2组,H2O2＋CR组,CR组和正常对照组 MTT法检测不同浓度H2O2作用下CR对细胞生存率的影响 TUNEL染色法检测过氧化氢作用后CR对细胞的凋亡的影响 免疫荧光检测PC12细胞中SIRT3的表达定位 RT-PCR及Western印迹检测SIRT3、Caspase-3的表达变化.结果 60μmol/L H2O2作用6 h后,H2O2组细胞生存率（74.01±2.21）%可维持在70%以上,与对照组比较差异有统计学意义（P＜0.05） 120μmol/L H2O2作用下,H2O2组的细胞活力显著下降（38.22±3.34）%,后续研究选取60μmol/L H2O2浓度作为应激源 60μmol/L H2O2作用6 h后H2O2组细胞生存率（74.01±2.21）%与CR＋H2O2组（97.26±1.92）%间比较差异有统计学意义（P＜0.05）.TUNEL凋亡检测H2O2＋CR组凋亡率较H2O2组显著降低.免疫荧光双染色证实PC12细胞中SIRT3为一种线粒体蛋白.Western印迹显示与正常对照组（5256±144）比较,CR组中SIRT3表达升高（6857±157）,差异有统计学意义（P＜0.05）、H2O2组中表达降低（3786±160）,差异有统计学意义（P＜0.05）.与H2O2组（3786±160）比较,CR＋H2O2组中SIRT3表达上升（5056±121）,差异有统计学意义（P＜0.05）.与正常对照组比较（5342±420）,H2O2组中Caspase-3表达升高（8499±426）,差异有统计学意义（P＜0.001）.CR＋H2O2组（5750±438）中Caspase-3表达较H2O2组表达下降（8499±426）,差异有统计学意义（P＜0.001）.RT-PCR显示与正常对照组（6204±134）比较,CR组（7214±148）中SIRT3表达升高,差异有统计学意义（P＜0.05）、H2O2组中表达降低（4807±143）,差异有统计学意义（P＜0.05）.与H2O2组（4807±143）比较,CR＋H2O2组中SIRT3表达上升（6195±166）,差异有统计学意义（P＜0.05）.结论 PC12细胞中,CR具有抗氧化应激损伤及凋亡的效应 CR可上调PC12细胞中SIRT3的表达,在H2O2诱导的PC12应激损伤�Objective To study the regulation effects of CR （caloric restriction） and SIRT3 in the H2O2-induced oxidative stress injury of PC12 cell. Methods The cells were divided into four groups：H2O2, H2O2＋CR, CR and control （high glucose）. For control and H2O2 group, cells were cultured in DMEM containing 0.45% glucose for group in CR condition, cells were treated with the medium containing 0.1% glucose. For groups with H2O2, the H2O2 was diluted in EMEM medium to obtain the final concentration containing 60 μmol/L. Viability of PC12 cells were measured by MTT assay. The medium was refreshed with different concentration of H2O2 （from 10 to 120 μmol/L）. The absorbance of the samples was measured at 492 nm using a microtiter plate reader. We detected TUNEL-positive cells using the In Situ Cell Apoptosis Detection kit pretreated with CR and H2O2. Immunofluorescence double staining detected the expression and localization of SIRT3. RT-PCR and Western-blot mehtods detected the expression of SIRT3,Caspase-3. Results After pretreating with 60 μmol/L H2O2 for 6 h, the viability of PC12 cells in H2O2 group （74.01±2.21）% retained above 70%, and have statistical significance contrasted with control group （P〈0.05） After pretreating with 120μmol/L H2O2, the viability of PC12 cells declined significantly （38.22±3.34）%. So 60μmol/L H2O2 is our experiment concentration . The viability of H2O2 group （74.01±2.21）% was much lower than CR ＋ H2O2 group （97.26±1.92）% （P〈0.05）. After pretreating with H2O2, TUNEL staining showed the apoptosis cells of CR＋H2O2 group decreased significantly contrasted with H2O2 group. The immunofluorescence double staining results showed that SIRT3was a mitochondria protein. Western-blot showed the expression of SIRT3 in CR group （6857±157） （P〈0.05） increased and decreased in H2O2 group （3786±160） （P〈0.05） contrasted with control group （5256±143）. The expression of SIRT3 in CR ＋ H2O2 group（5056±121）（P〈0.05）increased

关 键 词：氧化性应激 PC12细胞 过氧化氢 热量限制 

分 类 号：D919[医药卫生—法医学]