【摘要】：2型糖尿病和糖耐量受損是全世界范圍內(nèi)威脅人類健康的一種流行性疾病,其中胰島素抵抗是2型糖尿病的前奏。越來越多的實(shí)驗(yàn)證實(shí)硒對(duì)人體健康具有兩面性,缺硒和硒過量均會(huì)導(dǎo)致糖尿病的發(fā)病。硒蛋白S(SelS)是與糖尿病發(fā)生緊密相關(guān)的硒蛋白,屬于葡萄糖調(diào)節(jié)蛋白。大量游離脂肪酸FFA可以通過異位脂質(zhì)沉積和促進(jìn)炎癥反應(yīng)誘發(fā)胰島素抵抗。因此,探究SelS基因表達(dá)與胰島素抵抗之間的關(guān)聯(lián)對(duì)于2型糖尿病的研究具有潛在的應(yīng)用價(jià)值。本文以HepG2細(xì)胞作為研究對(duì)象,加入0.25mM軟脂酸Palmitate作用24h后,胰島素作用15min,從而探究SelS基因沉默前后細(xì)胞中糖異生作用和糖原合成的變化,以及胰島素信號(hào)通路中蛋白分子的水平變化。此外之外,重點(diǎn)探究了SelS基因沉默和腺苷酸活化蛋白激酶AMPK活性之間的關(guān)系和作用機(jī)理。我們采用RNA干擾技術(shù)、RT-PCR技術(shù)、Western Blotting、糖原試劑盒、分光光度法等方法,研究了SelS基因沉默對(duì)軟脂酸作用下細(xì)胞內(nèi)糖代謝效應(yīng)的作用及其機(jī)制。結(jié)果表明SelS基因沉默后,軟脂酸誘導(dǎo)下的糖異生相關(guān)基因表達(dá)顯著升高,糖原含量無明顯變化,信號(hào)途徑中的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,數(shù)據(jù)表明SelS基因沉默后胰島素敏感性得到了增強(qiáng)。同時(shí)SelS基因沉默下AMPK的磷酸化水平顯著升高,AMPK活性被大量激活。加入其抑制劑Compound C(CC)以后,SelS基因沉默下被抑制的糖異生基因表達(dá)明顯升高,胰島素抵抗加劇,表明SelS沉默下通過激活A(yù)MPK抑制了糖異生作用。我們的實(shí)驗(yàn)結(jié)果證實(shí)了SelS基因沉默通過激活A(yù)MPK緩解軟脂酸誘導(dǎo)下的胰島素抵抗,增強(qiáng)了胰島素信號(hào)通路的轉(zhuǎn)導(dǎo),改善了葡萄糖的代謝效應(yīng)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.1

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