SelS基因沉默对软脂酸诱导HepG2细胞胰岛素抵抗的作用及机制研究

发布时间：2018-07-23 10:31

【摘要】：2型糖尿病和糖耐量受损是全世界范围内威胁人类健康的一种流行性疾病,其中胰岛素抵抗是2型糖尿病的前奏。越来越多的实验证实硒对人体健康具有两面性,缺硒和硒过量均会导致糖尿病的发病。硒蛋白S(SelS)是与糖尿病发生紧密相关的硒蛋白,属于葡萄糖调节蛋白。大量游离脂肪酸FFA可以通过异位脂质沉积和促进炎症反应诱发胰岛素抵抗。因此,探究SelS基因表达与胰岛素抵抗之间的关联对于2型糖尿病的研究具有潜在的应用价值。本文以HepG2细胞作为研究对象,加入0.25mM软脂酸Palmitate作用24h后,胰岛素作用15min,从而探究SelS基因沉默前后细胞中糖异生作用和糖原合成的变化,以及胰岛素信号通路中蛋白分子的水平变化。此外之外,重点探究了SelS基因沉默和腺苷酸活化蛋白激酶AMPK活性之间的关系和作用机理。我们采用RNA干扰技术、RT-PCR技术、Western Blotting、糖原试剂盒、分光光度法等方法,研究了SelS基因沉默对软脂酸作用下细胞内糖代谢效应的作用及其机制。结果表明SelS基因沉默后,软脂酸诱导下的糖异生相关基因表达显著升高,糖原含量无明显变化,信号途径中的Akt,FOXO1和IRS-1的蛋白磷酸化水平升高,JNK磷酸化水平有所下降,数据表明SelS基因沉默后胰岛素敏感性得到了增强。同时SelS基因沉默下AMPK的磷酸化水平显著升高,AMPK活性被大量激活。加入其抑制剂Compound C(CC)以后,SelS基因沉默下被抑制的糖异生基因表达明显升高,胰岛素抵抗加剧,表明SelS沉默下通过激活AMPK抑制了糖异生作用。我们的实验结果证实了SelS基因沉默通过激活AMPK缓解软脂酸诱导下的胰岛素抵抗,增强了胰岛素信号通路的转导,改善了葡萄糖的代谢效应。
[Abstract]:Type 2 diabetes mellitus and impaired glucose tolerance are a worldwide epidemic disease threatening human health, in which insulin resistance is a prelude to type 2 diabetes. More and more experiments have proved that selenium has dual effects on human health. Selenium deficiency and excess selenium will lead to diabetes. Selenoprotein S (SelS) is a glucose-regulated protein, which is closely related to diabetes mellitus. FFA can induce insulin resistance through ectopic lipid deposition and inflammatory response. Therefore, exploring the association between SelS gene expression and insulin resistance has potential application value in type 2 diabetes. In this study, HepG2 cells were treated with 0.25 mm palmitate for 24 hours and insulin for 15 min, so as to explore the changes of glycosylation and glycogen synthesis before and after SelS gene silencing. And the changes of protein molecules in insulin signaling pathway. In addition, the relationship between SelS gene silencing and adenylate activated protein kinase (AMPK) activity was investigated. The effects of SelS gene silencing on intracellular glucose metabolism induced by palmitic acid were studied by RT-PCR, glycogen kit and spectrophotometry. The results showed that after the silencing of SelS gene, the expression of glycosylated allogeneic genes induced by palmitate was significantly increased, the glycogen content was not changed, and the protein phosphorylation level of Aktfon FOXO1 and IRS-1 in the signal pathway was increased and the level of JNK phosphorylation was decreased. The data showed that insulin sensitivity was enhanced after SelS gene silencing. At the same time, the phosphorylation level of AMPK was significantly increased under the silencing of SelS gene. After the addition of its inhibitor compound C (CC), the expression of glycosylated allogeneic gene inhibited by SelS gene silencing was significantly increased, and insulin resistance was aggravated, which indicated that SelS silencing inhibited the glycosylation by activating AMPK. Our results confirmed that SelS gene silencing alleviates insulin resistance induced by palmitate by activating AMPK, enhances insulin signal transduction and improves glucose metabolism.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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