衰老过程中线粒体顺乌头酸酶活性变化对能量合成的影响

发布时间：2018-05-24 15:23

本文选题：衰老 + 线粒体　；参考：《中国病理生理杂志》2013年07期

【摘要】：目的:利用自然衰老雄性SD大鼠和D-半乳糖(D-Gal)诱导的细胞衰老模型,研究在衰老细胞中线粒体顺乌头酸酶(mitochondrial aconitase,ACO2)活性的变化及其对能量合成的影响。方法:利用55 mmol/L D-Gal诱导人胚肺成纤维细胞MRC-5衰老,并采用蔗糖连续密度梯度离心法分离大鼠脑组织和MRC-5细胞的线粒体;ACO2活性根据反应产物NADPH在340 nm的吸光度变化率进行检测,其表达水平分析分别用荧光定量PCR和Western blotting完成;利用试剂盒检测细胞超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量以反映细胞氧化还原状态;组织铁含量的检测则通过菲咯嗪法进行;线粒体膜电位及腺苷酸含量分别应用JC-1荧光探针法和高效液相色谱法检测,并计算能荷。结果:随着大鼠月龄增加,ACO2活性逐渐下降,表达水平无明显差异,组织铁水平逐渐升高;体外氧化应激实验显示ACO2活性随H2O2处理浓度和时间增加而下降;细胞衰老模型中,SOD活性降低,MDA含量升高,ACO2活性下降而表达水平未变;自然衰老大鼠和细胞衰老模型中,线粒体膜电位、ATP合成与能荷呈明显下降趋势。结论:衰老过程中,随着氧化压力增加,氧化应激敏感的ACO2活性下降,影响三羧酸循环效率,同时伴线粒体膜电位下降,进而使得能量合成降低。
[Abstract]:Objective: To study the changes of mitochondrial CIS (mitochondrial aconitase, ACO2) activity in aging cells and the effect on the energy synthesis in aging SD rats and D- galactose (D-Gal). Methods: using 55 mmol/L D-Gal to induce the MRC-5 senescence of human embryonic lung fibroblasts and use sucrose. The mitochondria of rat brain tissue and MRC-5 cells were separated by continuous density gradient centrifugation; ACO2 activity was detected by the reaction product NADPH at 340 nm absorbance rate, and the expression level analysis was performed by fluorescence quantitative PCR and Western blotting, respectively. The activity of cell superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by the reagent box. The content of the cells was redox state, and the content of the tissue was detected by the phenanthrazine method. The mitochondrial membrane potential and adenylate content were detected by JC-1 fluorescence probe and high performance liquid chromatography, and the energy charge was calculated. The results showed that with the increase of the age of the rats, the activity of ACO2 gradually decreased and the expression level was not significantly different. In vitro oxidative stress experiments showed that the activity of ACO2 decreased with the increase of concentration and time of H2O2 treatment. In the cell aging model, the activity of SOD decreased, the content of MDA increased, the activity of ACO2 decreased and the expression level was not changed. In the natural aging rats and cell aging models, the grain potential of the grain body, the synthesis of ATP and the energy charge were obviously decreased. Conclusion: during the aging process, the oxidative stress sensitive ACO2 activity decreased with the increase of oxidative stress, which affected the efficiency of the three carboxylic acid cycle, while the mitochondrial membrane potential decreased, which reduced the energy synthesis.
【作者单位】：温州医学院检验医学与生命科学学院;温州医学院环境与公共卫生学院;
【基金】：国家973计划项目(No.2007CB507402) 浙江省自然科学基金资助项目(No.Y2100582) 浙江省教育厅科研基金资助项目(No.Y200804470)
【分类号】：R363

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