【作者】 李鹏；

【导师】 钟才高；

【作者基本信息】 中南大学 ， 公共卫生与预防医学， 2012， 硕士

【摘要】 目的：在体外试验系统,初步探讨Cr(Ⅵ)诱导线粒体DNA氧化损伤情况及hOGGl基因的修复作用,为进一步阐明Cr(Ⅵ)诱导线粒体氧化损伤的机制提供线索。方法：以L-02肝细胞为受试细胞,通过MTT试验检测不同浓度Cr(Ⅵ)对L-02肝细胞存活率的影响,筛选出Cr(Ⅵ)浓度高(32umol/L)、中(8μmol/L)、低(2μmol/L)和空白对照(0μmol/L)进行后续试验,染毒时间为24h。通过多功能荧光酶标仪检测细胞内活性氧(ROS)及ATP的浓度,实时荧光定量PCR(RT-qPCR)检测hOGG1基因mRNA的表达,酶标仪检测线粒体内8-羟基脱氧鸟苷(8-OHdG)的含量及细胞内超氧化物歧化酶(SOD)活性,Western blot方法检测线粒体内8-羟基鸟嘌呤DNA糖苷酶(hOGG1蛋白)含量,分光光度计检测细胞内过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)活性。结果：1.Cr(Ⅵ)引起L-02肝细胞存活率降低：Cr(Ⅵ)在2-256μmol/L处理浓度范围内(处理24h),能明显引起L-02肝细胞存活率的降低(P<0.05),处理浓度和细胞存活率之间存在明显负相关(r=-0.924,P<0.01)。根据细胞存活率选出适当的Cr(Ⅵ)处理浓度：高(32μmol/L)、中(8μmol/L)、低(2μmol/L)和空白对照(0μmol/L)进行后续试验。2.Cr(Ⅵ)引起L-02肝细胞活性氧簇(ROS)含量增加：与对照组比较,2μmol/L Cr(Ⅵ)处理浓度组细胞内ROS平均含量无明显增加(p>0.05),8、32μmol/L Cr(Ⅵ)处理组细胞内ROS平均含量逐渐增加(p<0.05),且随着Cr(Ⅵ)处理浓度的增大,细胞内ROS平均含量随之增加,两者之间存在明显正相关(r=0.942,P<0.01)。3.Cr(Ⅵ)对L-02肝细胞能量代谢的影响：与对照组相比,2μmol/LCr(Ⅵ)低浓度组细胞内ATP浓度无变化(p>0.05);8、32μmol/LCr(Ⅵ)浓度组细胞内ATP浓度明显降低(P<0.05),且随着Cr(Ⅵ)处理浓度的增大,细胞内ATP浓度随之降低,两者之间存在明显负相关(r=-0.967,P<0.05)。4.Cr(Ⅵ)对线粒体内8-OHdG含量的影响：与对照组相比,2μmol/L Cr(VI)低浓度组线粒体内8-OHdG平均含量无变化(p>0.05)；8、32μmol/LCr(VI)浓度组线粒体内8-OHdG平均含量明显降低(P<0.05),且随着Cr(Ⅵ)处理浓度的增大,线粒体内8-OHdG含量随之增加,两者之间存在明显正相关(r=0.816,P<0.05)。5.Cr(Ⅵ)对hOGG1基因表达的影响：与对照组相比,2μmol/L处理组hOGG1基因mRNA表达水平及线粒体内hOGG1蛋白含量均明显增加(p<0.05)；8μmol/L浓度组hOGG1基因mRNA表达水平及线粒体内hOGG1蛋白含量与对照组相比无明显改变(p>0.05),但低于2μmol/L处理组(p<0.05);32μmol/L浓度组两者均明显降低(p<0.05),且在2-32μmol/L Cr(VI)处理浓度范围内,hOGG1基因mRNA表达水平及线粒体内hOGG1蛋白含量逐渐降低。6.Cr(Ⅵ)对L-02肝细胞抗氧化酶活力的影响：与对照组相比,2μmol/L浓度组细胞内SOD、CAT和GSH-PX平均活力明显增加(p<0.01);8μmol/L浓度组细胞内SOD和CAT平均活力明显降低(p<0.01),细胞内GSH-PX平均活力明显升高,且高于2μmol/L浓度组(p<0.01);32μmol/LCr(VI)浓度组细胞内SOD、CAT和GSH-PX平均活力均明显降低。结论：Cr(Ⅵ)可诱导细胞内ROS水平增加,使mtDNA受到氧化损伤,线粒体内8-OHdG含量增加,影响ATP的产生,导致细胞发生能量代谢障碍。而hOGG1基因表达水平及抗氧化酶活力(SOD、CAT和GSH-PX)的改变,影响了线粒体DNA的修复。总之,hOGG1基因在Cr(Ⅵ)诱导线粒体DNA氧化损伤中起到了重要的作用。更多还原

【Abstract】 Objective:To preliminarily explore mitochondria DNA oxidative damage and the repair effect of human8-hydroxyguanine deoxyribonucleic acid glycosidase (hOGG1) in Cr(Ⅵ)-treated hepatocytes in vitro, and to provide novel clues for further elucidating the mechanism of Cr(Ⅵ)-induced mitochondria oxidative damage.Methods:L-02human hepatocyte was used in this study. By applying MTT method, we detected the effect of Cr(Ⅵ) on hepatocytes survival rate and then chose four concentrations (0,2,8,32μmol/L) with the exposure time of24h for the following study. Cellular ROS, ATP levels as well as mitochondrial8-hydroxydeoxy guano sine (8-OHdG) content and superoxide dismutase (SOD) activity were measured by the multifunctional fluorescence microplate reader; the mRNA expression level of hOGG1was detected by real-time quantitative polymerase chain reaction (RT-qPCR); mitochondrial hOGGl protein expression level was evaluated by western blotting; and the activities of catalase (CAT) and glutathione peroxidase (GSH-PX) were determined by spectrophotometer.Results:1. Cr(Ⅵ) induces the inhibition of L-02hepatocytes survival rate. In the concentration range of2-256μmol/L, Cr(Ⅵ) significantly inhibited the hepatocytes survival rate (p<0,05), and a obvious negative correlation was found between the Cr(Ⅵ)-treated concentrations and cell survival rate (r=-0.924, p<0.05). Three suitable Cr(Ⅵ)-treated concentrations32,8, and2μmol/L (high, moderate and low) were chosen for the following studies based on the MTT assay.2. Cr(Ⅵ) induces ROS accumulation in L-02hepatocytes. Compared with the control group, the2μmol/L treatment group showed no significant increase of ROS levels (p>0.05), while the8,32μmol/L treatment group showed great increase of ROS levels (p<0.05) in a dose-response manner. Cr(Ⅵ)-treated concentrations and cellular ROS levels were positively correlated (r=0.942,;?<0.05).3. The effect of Cr(Ⅵ) on energy metabolism of L-02hepatocytes. Compared with control, the2μmol/L treatment group showed the similar ATP levels (p>0.05). The8,32μmol/L treatment group showed decreased ATP levels (P<0.05) in a dose-response manner. Cr(Ⅵ)-treated concentrations and ATP levels were negatively correlated (r=-0.967, p<0.05).4. The effect of Cr(Ⅵ) on mitochondrial DNA (mtDNA). Compared with control, the2μmol/L treatment group showed no significant change of8-OHdG content (p>0.05). The8,32μmol/L treatment group showed increased8-OHdG content (P<0.05) in a dose-response manner. Cr(Ⅵ)-treated concentrations and8-OHdG content were positively correlated (r=0.816,p<0.05).5. The effect of Cr(Ⅵ) on the expression of hOGGl. Compared with that of control, the levels of both mRNA and protein in the2μmol/L treatment group were significantly increased (P<0.05); the levels in the8μmol/L treatment group showed no significant change (p>0.05), and lower than that of the2μmol/L treatment group (P<0.05); the levels in the32μmol/L treatment group were significantly decreased (P<0.05). In the concentration range of2-32μmol/L, the levels of both mRNA and protein were gradually decreased.6. The effect of Cr(Ⅵ) on the enzymes of antioxidant system. Compared with that of control, the activities of SOD, CAT and GSH-PX were all significantly increased in the2μmol/L treatment group (P<0.01); the activities of SOD and CAT were decreased in the8μmol/L treatment group, while the activity of GSH-PX was increased, and higher than that of the2μmol/L treatment group (P<0.05); the activities of all of the enzymes in the32μmol/L treatment group were significantly decreased (P<0.05).Conclusions:Cr(Ⅵ) induces ROS accumulation in the hepatocytes, which further damages mtDNA, increases mitochondrial8-OHdG content, affects ATP production, causes energy metabolism dysfunction. The changes of hOGG1mRNA level and enzymes activities of antioxidant system affect the repair ability of mtDNA. In a word, hOGGl plays an important role in Cr(Ⅵ)-induced mtDNA oxidative damage.更多还原