亚硒酸钠对超氧阴离子诱导血管平滑肌细胞向成骨细胞分化作用的影响及机理

发布时间：2018-03-03 00:09

本文选题：亚硒酸钠　切入点：超氧阴离子　出处：《华中科技大学》2011年硕士论文　论文类型：学位论文

【摘要】：心血管疾病包括动脉粥样硬化,冠状动脉疾病等,是人类健康的重要危险因素。血管钙化是心血管疾病的共同病理特征,血管平滑肌细胞向成骨细胞分化是血管钙化的细胞学基础。血管钙化是一个类似于骨形成的、主动的和复杂的过程。研究表明微量元素硒的摄入量与癌症的发生率和死亡率呈现负相关性,提示硒有抗癌作用,而硒对心血管疾病的作用及其可能的机制尚不清楚。硒有清除自由基的作用。外源性的过氧化氢和超氧阴离子促进血管钙化。本文采用文献方法获取血管平滑肌细胞的体外钙化模型,通过黄嘌呤/黄嘌呤氧化酶体系产生超氧阴离子,研究亚硒酸钠是否抑制超氧阴离子诱导的血管平滑肌细胞向成骨细胞的分化以及可能的机制。 ⒈MTT法检测不同浓度的黄嘌呤(X)/黄嘌呤氧化酶(XO)体系对细胞活力的影响,检测超氧阴离子对血管平滑肌细胞向成骨细胞分化的标记蛋白碱性磷酸酶的活性的影响,原子吸收光谱测定细胞的基质钙沉积量。结果显示:低浓度的超氧阴离子对细胞活力,ALP活性和基质钙沉积量无显著影响;中浓度的超氧阴离子对细胞活力影响不显著,但抑制ALP活性和基质钙沉积;高浓度的超氧阴离子对细胞活力影响较大,对细胞毒性大,促进了ALP活性和基质钙沉积。 ⒉钙化的VSMCs加入亚硒酸钠预处理24h,MAPKK抑制剂PD98059预处理2h,X(μmol/L )/XO(mU/ml)(即浓度为100/40和100/80)作用一定时间后,MTT法检测细胞活力和检测细胞ALP活性,研究亚硒酸钠是否对超氧阴离子诱导的血管平滑肌细胞向成骨细胞的分化有抑制作用。结果表明:加X/XO作用3天时,亚硒酸钠对黄嘌呤/黄嘌呤氧化酶体系引起的细胞毒性有显著的抑制作用,而作用7天时,亚硒酸钠的抑制作用不明显;亚硒酸钠对超氧阴离子诱导的ALP活性的影响有一定的抑制作用。 ⒊研究亚硒酸钠抑制超氧阴离子诱导的VSMCs向成骨细胞分化的可能涉及的机理。检测细胞谷胱甘肽过氧化酶的活性和观察胞内活性氧物种(ROS)水平,Westernblotting分析磷酸化的ERK水平,研究亚硒酸钠和超氧阴离子对MAPK信号转导途径中磷酸化的ERK的活性的影响。结果表明:超氧阴离子降低了细胞的GPx的活性,而加入亚硒酸钠,可增加细胞的GPx的活性,表明亚硒酸钠通过上调GPx活性有抑制细胞氧化损伤的作用;与对照组相比较,X/XO组和亚硒酸钠预处理的X/XO组的胞内ROS水平有一定程度的增加,但亚硒酸钠作用不显著;亚硒酸钠对超氧阴离子激活的磷酸化的ERK水平有抑制作用,加入MAPKK抑制剂PD98059,可以抑制超氧阴离子激活的磷酸化的ERK的水平,提示亚硒酸钠可能通过MAPK途径抑制超氧阴离子诱导的血管平滑肌细胞向成骨细胞的分化。
[Abstract]:Cardiovascular diseases, including atherosclerosis and coronary artery disease, are important risk factors for human health. Vascular calcification is a common pathological feature of cardiovascular disease. The differentiation of vascular smooth muscle cells into osteoblasts is the cellular basis of vascular calcification, which is similar to bone formation. Active and complex processes. Studies have shown a negative correlation between the intake of trace element selenium and the incidence and mortality of cancer, suggesting that selenium has anticancer effects. The effect of selenium on cardiovascular disease and its possible mechanism are unclear. Selenium can scavenge free radicals. Exogenous hydrogen peroxide and superoxide anion can promote vascular calcification. The calcification model of vascular smooth muscle cells in vitro was obtained by using literature method. Superoxide anion was produced by xanthine / xanthine oxidase system. To investigate whether sodium selenite inhibits the differentiation of vascular smooth muscle cells induced by superoxide anion into osteoblasts and its possible mechanism. 1MTT assay was used to detect the effects of xanthine xanthine / xanthine oxidase (XO) at different concentrations on cell viability, and the effect of superoxide anion on the activity of alkaline phosphatase (ALP), a marker protein in the differentiation of vascular smooth muscle cells (VSMC) into osteoblasts. The results showed that the low concentration of superoxide anion had no significant effect on the activity of ALP and the amount of calcium deposition in the matrix, but the medium concentration of superoxide anion had no significant effect on the cell viability. High concentration of superoxide anion had great effect on cell viability and cytotoxicity, and promoted ALP activity and matrix calcium deposition. 2 the calcified VSMCs was pretreated with sodium selenite for 24 h, and then pretreated with PD98059 for 2 h (渭 mol/L mol/L / XOU / U / ml) (100/40 and 100 / 80) for a certain period of time, then the cell viability and the activity of ALP were detected by MTT assay. To study whether sodium selenite can inhibit the differentiation of vascular smooth muscle cells into osteoblasts induced by superoxide anion. Sodium selenite significantly inhibited cytotoxicity induced by xanthine / xanthine oxidase system. Sodium selenite inhibited the activity of ALP induced by superoxide anion. 3 the possible mechanism of sodium selenite inhibiting the differentiation of VSMCs into osteoblasts induced by superoxide anion was studied. The activity of glutathione peroxidase (GSH) and intracellular reactive oxygen species (Ros) were measured. Western blotting was used to analyze the phosphorylation level of ERK. The effects of sodium selenite and superoxide anion on the activity of phosphorylated ERK in MAPK signal transduction pathway were studied. The results showed that superoxide anion decreased the activity of GPx, and sodium selenite increased the activity of GPx. The results showed that sodium selenite inhibited cell oxidative damage by up-regulating the activity of GPx, but sodium selenite had no significant effect on the intracellular ROS level in X- / XO group and X- / XO group, but sodium selenite had no significant effect compared with the control group. Sodium selenite inhibited the level of phosphorylated ERK activated by superoxide anion. The addition of MAPKK inhibitor PD98059 could inhibit the level of phosphorylated ERK activated by superoxide anion. It is suggested that sodium selenite may inhibit the differentiation of vascular smooth muscle cells induced by superoxide anion into osteoblasts through MAPK pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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