亚硒酸钠诱导HepG2细胞胰岛素抵抗的作用及机制研究

发布时间：2018-05-24 05:56

本文选题：2型糖尿病 + 胰岛素抵抗　；参考：《华中科技大学》2015年硕士论文

【摘要】：硒是哺乳动物的一种必需微量元素。研究表明,硒具有类胰岛素作用;而近来越来越多的研究表明,缺硒和硒过量会促进糖尿病的发生发展,但内在的机理仍然知之甚少。胰岛素抵抗在2型糖尿病的发病机理中极其重要。因此,硒与胰岛素抵抗之间关系的研究对深入了解硒与糖尿病的关系具有重要意义,对预防和治疗2型糖尿病具有潜在的应用价值。本文以亚硒酸钠Na2SeO3处理的HepG2细胞为研究对象,研究了亚硒酸钠对胰岛素效应与信号通路的影响及其机制,重点探讨了高浓度亚硒酸钠(1μM,2.5μM和5μM)对HepG2细胞的糖代谢(包括糖原合成和糖异生)、肝脏细胞中胰岛素信号通路、氧化应激水平、内质网应激水平的影响,主要结果如下:我们采用糖原试剂盒、实时荧光定量PCR技术、蛋白质印迹技术、分光光度法、活性氧试剂盒,研究了亚硒酸钠对HepG2细胞胰岛素信号通路的影响及可能的机制。结果表明,高浓度亚硒酸钠使得HepG2细胞内糖原含量降低,促进了糖异生基因的表达,降低了Akt的磷酸化水平。这说明高浓度亚硒酸钠导致胰岛素敏感性降低,抑制胰岛素信号传导,引起胰岛素抵抗。亚硒酸钠的加入没有改变GRP78的表达,说明在HepG2细胞中,亚硒酸钠可能不是或主要不是通过内质网应激引起胰岛素抵抗的。高硒处理使细胞中ROS含量增加,加入抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)可以降低高硒引起的ROS水平升高。NAC的加入可以逆转由亚硒酸钠导致的糖原合成减少和糖异生基因表达增加,这说明亚硒酸钠是通过ROS作用于胰岛素信号通路的。同时NAC的加入使得Akt磷酸化水平升高,JNK磷酸化水平降低,IRS1酪氨酸磷酸化水平升高,说明ROS的减少能够增强胰岛素信号,ROS通过激活JNK而导致IRS1酪氨酸磷酸化降低而抑制胰岛素信号转导,引起胰岛素抵抗,抑制糖原合成,促进糖异生。我们的实验结果表明,亚硒酸钠通过诱导HepG2细胞中产生ROS,激活JNK,通过抑制IRS1的酪氨酸磷酸化而抑制胰岛素信号转导,引起胰岛素抵抗。
[Abstract]:Selenium is an essential trace element in mammals. Studies have shown that selenium has insulin-like effects, and more recent studies have shown that selenium deficiency and excess selenium can promote the development of diabetes, but the underlying mechanism is still poorly understood. Insulin resistance is very important in the pathogenesis of type 2 diabetes. Therefore, the study on the relationship between selenium and insulin resistance is of great significance in understanding the relationship between selenium and diabetes mellitus, and has potential application value in the prevention and treatment of type 2 diabetes mellitus. The effect of sodium selenite on insulin effect and signal pathway and its mechanism were studied in HepG2 cells treated with sodium selenite Na2SeO3. The effects of high concentration of sodium selenite (1 渭 M, 2.5 渭 M and 5 渭 M) on glucose metabolism (including glycogen synthesis and glycosylation, insulin signaling pathway, oxidative stress level, endoplasmic reticulum stress level) in HepG2 cells were studied. The main results are as follows: the effects of sodium selenite on insulin signaling pathway in HepG2 cells were studied by using glycogen kit real-time fluorescence quantitative PCR Western blot spectrophotometry and reactive oxygen species kit. The results showed that high concentration of sodium selenite reduced the content of glycogen in HepG2 cells, promoted the expression of glycosylated allogenes and reduced the phosphorylation level of Akt. This suggests that high concentration of sodium selenite leads to decrease of insulin sensitivity, inhibition of insulin signal transduction and insulin resistance. The addition of sodium selenite did not change the expression of GRP78, suggesting that sodium selenite may not or mainly not induce insulin resistance in HepG2 cells through endoplasmic reticulum stress. High selenium treatment increased the content of ROS in the cells. The addition of the antioxidant N-acetylcysteine NACcould reduce the increase of ROS level induced by high selenium. The addition of NAC could reverse the decrease of glycogen synthesis induced by sodium selenite and the increase of glycosylcysteine gene expression. This suggests that sodium selenite acts on the insulin signaling pathway through ROS. At the same time, the addition of NAC increased the level of Akt phosphorylation and decreased the level of tyrosine phosphorylation of IRS1. It is suggested that the decrease of ROS can enhance the insulin signal. Ros can inhibit the insulin signal transduction by activating JNK and leading to the decrease of IRS1 tyrosine phosphorylation, which can induce insulin resistance, inhibit glycogen synthesis and promote glycosylation. Our results show that sodium selenite induces ROS in HepG2 cells, activates IRS1 and inhibits insulin signal transduction and insulin resistance by inhibiting tyrosine phosphorylation of IRS1.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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