本文選題：亞硒酸鈉　切入點(diǎn)：超氧陰離子　出處：《華中科技大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：心血管疾病包括動脈粥樣硬化,冠狀動脈疾病等,是人類健康的重要危險因素。血管鈣化是心血管疾病的共同病理特征,血管平滑肌細(xì)胞向成骨細(xì)胞分化是血管鈣化的細(xì)胞學(xué)基礎(chǔ)。血管鈣化是一個類似于骨形成的、主動的和復(fù)雜的過程。研究表明微量元素硒的攝入量與癌癥的發(fā)生率和死亡率呈現(xiàn)負(fù)相關(guān)性,提示硒有抗癌作用,而硒對心血管疾病的作用及其可能的機(jī)制尚不清楚。硒有清除自由基的作用。外源性的過氧化氫和超氧陰離子促進(jìn)血管鈣化。 本文采用文獻(xiàn)方法獲取血管平滑肌細(xì)胞的體外鈣化模型,通過黃嘌呤/黃嘌呤氧化酶體系產(chǎn)生超氧陰離子,研究亞硒酸鈉是否抑制超氧陰離子誘導(dǎo)的血管平滑肌細(xì)胞向成骨細(xì)胞的分化以及可能的機(jī)制。 ⒈MTT法檢測不同濃度的黃嘌呤(X)/黃嘌呤氧化酶(XO)體系對細(xì)胞活力的影響,檢測超氧陰離子對血管平滑肌細(xì)胞向成骨細(xì)胞分化的標(biāo)記蛋白堿性磷酸酶的活性的影響,原子吸收光譜測定細(xì)胞的基質(zhì)鈣沉積量。結(jié)果顯示:低濃度的超氧陰離子對細(xì)胞活力,ALP活性和基質(zhì)鈣沉積量無顯著影響;中濃度的超氧陰離子對細(xì)胞活力影響不顯著,但抑制ALP活性和基質(zhì)鈣沉積;高濃度的超氧陰離子對細(xì)胞活力影響較大,對細(xì)胞毒性大,促進(jìn)了ALP活性和基質(zhì)鈣沉積。 ⒉鈣化的VSMCs加入亞硒酸鈉預(yù)處理24h,MAPKK抑制劑PD98059預(yù)處理2h,X(μmol/L )/XO(mU/ml)(即濃度為100/40和100/80)作用一定時間后,MTT法檢測細(xì)胞活力和檢測細(xì)胞ALP活性,研究亞硒酸鈉是否對超氧陰離子誘導(dǎo)的血管平滑肌細(xì)胞向成骨細(xì)胞的分化有抑制作用。結(jié)果表明:加X/XO作用3天時,亞硒酸鈉對黃嘌呤/黃嘌呤氧化酶體系引起的細(xì)胞毒性有顯著的抑制作用,而作用7天時,亞硒酸鈉的抑制作用不明顯;亞硒酸鈉對超氧陰離子誘導(dǎo)的ALP活性的影響有一定的抑制作用。 ⒊研究亞硒酸鈉抑制超氧陰離子誘導(dǎo)的VSMCs向成骨細(xì)胞分化的可能涉及的機(jī)理。檢測細(xì)胞谷胱甘肽過氧化酶的活性和觀察胞內(nèi)活性氧物種(ROS)水平,Westernblotting分析磷酸化的ERK水平,研究亞硒酸鈉和超氧陰離子對MAPK信號轉(zhuǎn)導(dǎo)途徑中磷酸化的ERK的活性的影響。結(jié)果表明:超氧陰離子降低了細(xì)胞的GPx的活性,而加入亞硒酸鈉,可增加細(xì)胞的GPx的活性,表明亞硒酸鈉通過上調(diào)GPx活性有抑制細(xì)胞氧化損傷的作用;與對照組相比較,X/XO組和亞硒酸鈉預(yù)處理的X/XO組的胞內(nèi)ROS水平有一定程度的增加,但亞硒酸鈉作用不顯著;亞硒酸鈉對超氧陰離子激活的磷酸化的ERK水平有抑制作用,加入MAPKK抑制劑PD98059,可以抑制超氧陰離子激活的磷酸化的ERK的水平,提示亞硒酸鈉可能通過MAPK途徑抑制超氧陰離子誘導(dǎo)的血管平滑肌細(xì)胞向成骨細(xì)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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