高表达Mn-SOD肝细胞模型的构建及应用

发布时间：2018-09-04 21:02

【摘要】： 活性氧物质(ROS)是生物体内氧的某些代谢产物和一些反应的含氧产物的总称,与细胞凋亡、细胞周期、信号转导,细胞分化、衰老等很多生理学现象密切相关,一般被认为是具有有害和有利双重作用的。过度的氧化应激将导致细胞的坏死,而适度的氧化应激则诱导细胞增殖。ROS处于一个动态平衡中是其行使正常功能所必须,其中超氧阴离子(O_2~-)是胞内氧化压力的重要来源,并能通过反应转化为其他各类ROS。作为活性氧的一种清除剂,超氧化物歧化酶能够催化超氧阴离子O2.-反应生成H_2O_2和水,在降低胞内O2.-水平和胞内氧化压力起到重要的作用,尤其是位于线粒体的Mn-SOD,是清除ROS第一道防线,几乎任何癌的Mn-SOD都明显低于正常,而且也有研究表明Mn-SOD可提高细胞对氧应激反应的耐受性。在本研究中,我们通过转染含有Mn-SOD的真核表达质粒进入肝细胞HepG2,构建出高Mn-SOD表达的肝细胞模型,证明Mn-SOD在双氧水的刺激下对线粒体活性存在保护作用,具有较高Mn-SOD水平的细胞相对Mn-SOD缺乏的细胞而言具有对双氧水的细胞毒性更好的抵抗作用。此外,我们用流式细胞仪检测转染Mn-SOD对细胞中ROS水平和细胞周期的影响,为了寻找Mn-SOD抗氧化作用的具体信号通路,我们用Western检测了Akt的磷酸化的状态,用RT-PCR检测了转录因子的表达水平。实验显示,Mn-SOD的高表达抑制Akt的磷酸化和降低氧化敏感转录因子c-Fos/c-Jun和p65的表达水平,这些结果表明Mn-SOD通过降低ROS水平,调节Akt的磷酸化从而抑制转录因子c-Fos/c-Jun and p65的表达,终致抗氧化能力的提高。
[Abstract]:Reactive oxygen species (ROS) is a general term for some metabolites of oxygen and some reactive oxygen products in organisms, which are closely related to many physiological phenomena, such as apoptosis, cell cycle, signal transduction, cell differentiation, senescence and so on. It is generally considered to have both harmful and beneficial effects. Excessive oxidative stress leads to cell necrosis, while moderate oxidative stress induces cell proliferation. Ros in a dynamic balance is necessary for its normal function, and superoxide anion is an important source of intracellular oxidative pressure. And can be converted into other kinds of ROS. by reaction. As a scavenger of reactive oxygen species, superoxide dismutase can catalyze the reaction of superoxide anion O 2.- to produce H_2O_2 and water, which plays an important role in reducing the level of intracellular O 2-and the intracellular oxidation pressure. In particular, Mn-SOD, located in mitochondria is the first line of defense to clear ROS, Mn-SOD in almost any cancer is significantly lower than normal, and some studies have shown that Mn-SOD can improve the tolerance of cells to oxygen stress. In this study, we constructed a hepatocyte model with high Mn-SOD expression by transfection of eukaryotic expression plasmid containing Mn-SOD into hepatocyte HepG2, which proved that Mn-SOD has protective effect on mitochondrial activity induced by hydrogen peroxide. The cells with higher Mn-SOD level had better resistance to the cytotoxicity of hydrogen peroxide than those lacking Mn-SOD. In addition, we used flow cytometry to detect the effect of Mn-SOD transfection on ROS level and cell cycle. In order to find the specific signal pathway of Mn-SOD antioxidation, we used Western to detect the phosphorylation of Akt. The expression of transcription factors was detected by RT-PCR. The results showed that the high expression of Mn-SOD inhibited the phosphorylation of Akt and the expression of oxidative sensitive transcription factors c-Fos/c-Jun and p65. These results indicated that Mn-SOD inhibited the expression of c-Fos/c-Jun and p65 by decreasing the level of ROS and regulating the phosphorylation of Akt. The ultimate increase in antioxidant capacity.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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