GBP5和HULC在炎癥誘導(dǎo)的胰島素抵抗中的作用機制研究
發(fā)布時間：2018-12-12 05:49
【摘要】：糖尿病是一種內(nèi)分泌代謝性疾病,其誘因是由于胰島素分泌不足,進(jìn)而導(dǎo)致血糖過高,最終引起蛋白質(zhì)和脂肪的代謝發(fā)生紊亂。目前,糖尿病嚴(yán)重威脅著人類的健康,2016年的世界衛(wèi)生組織調(diào)查顯示,目前我國成年人的糖尿病發(fā)病率已經(jīng)接近10%,形勢十分嚴(yán)峻。普遍研究表明,胰島素抵抗(insulin resistance,IR)是導(dǎo)致2型糖尿病的主要原因,在胰島素抵抗個體中,正常量的胰島素不能使脂肪、骨骼肌和肝臟從血液中攝取葡萄糖,血糖持續(xù)升高,使胰島代償性分泌更多的胰島素以維持血糖的穩(wěn)定,而胰島β細(xì)胞不能代償高血糖將導(dǎo)致代謝綜合征和2型糖尿病。本課題主要針對GBP5和HULC是否參與炎癥因子誘導(dǎo)的胰島素抵抗進(jìn)行研究。GBP5屬于鳥苷酸結(jié)合蛋白GBPs家族的一種,可以被細(xì)菌脂多糖(LPS)高誘導(dǎo)。另外,GBP5還能夠促進(jìn)哺乳動物NLRP3炎癥小體的組裝和免疫。而HULC屬于lncRNA的一種,目前對于lncRNA的新興的研究涉及到發(fā)育,分化,生長組織損傷、修復(fù)、再生,及代謝的廣泛的生物過程。新的疾病相關(guān)lncRNA基因也正在發(fā)現(xiàn)當(dāng)中,并且其分子機制正在被一步一步發(fā)現(xiàn)和闡明。但是,GBP5和HULC的表達(dá)調(diào)控是否受炎癥因子調(diào)控還不清楚,并且,GBP5和HULC是否調(diào)節(jié)肝臟胰島素抵抗也還不清楚。為此,本課題針對炎癥因子是否影響GBP5和HULC的表達(dá)水平,以及GBP5和HULC是否調(diào)節(jié)肝臟炎癥誘導(dǎo)的胰島素抵抗進(jìn)行了探究。研究結(jié)果如下:第一部分:通過普通PCR驗證和Realtime PCR驗證,發(fā)現(xiàn)炎癥因子(TNF-α、IFN-γ混合物)處理Hepa1細(xì)胞后,可以促進(jìn)GBP5高表達(dá),且炎癥因子處理組比對照組GBP5表達(dá)水平高2200倍左右。根據(jù)以上實驗結(jié)果,采用TNF-α和IFN-γ單獨處理肝臟細(xì)胞,發(fā)現(xiàn)在Hepa1細(xì)胞中,TNF-α處理組GBP5的表達(dá)升高了1.7倍左右,IFN-γ處理組GBP5表達(dá)升高了700倍左右;而在肝實質(zhì)細(xì)胞中,TNF-α處理組GBP5的表達(dá)升高了5倍以上。此實驗證明炎癥因子可以促進(jìn)GBP5的高表達(dá)。那么GBP5是否參與了炎癥誘導(dǎo)的胰島素抵抗呢?為了探究這個問題,本課題構(gòu)建了GBP5過表達(dá)腺病毒,在原代肝實質(zhì)細(xì)胞中過表達(dá)后,檢測胰島素誘導(dǎo)的Akt磷酸化水平,進(jìn)而判斷GBP5是否參與炎癥誘導(dǎo)的胰島素抵抗。結(jié)果發(fā)現(xiàn)過表達(dá)GBP5后,Akt蛋白表達(dá)水平降低,Akt蛋白磷酸化水平降低,證明過表達(dá)GBP5可以參與炎癥誘導(dǎo)的胰島素抵抗。第二部分:通過普通PCR驗證和Realtime PCR驗證,發(fā)現(xiàn)炎癥因子TNF-α處理HepG2細(xì)胞后,可以促進(jìn)HULC高表達(dá),且炎癥因子處理組比無血清處理組的HULC表達(dá)水平高2.5倍左右。為了進(jìn)一步探究HULC是否參與了炎癥誘導(dǎo)的胰島素抵抗,本課題構(gòu)建了HULC過表達(dá)腺病毒,在HepG2細(xì)胞中過表達(dá)后,檢測胰島素誘導(dǎo)的Akt磷酸化水平,結(jié)果發(fā)現(xiàn)過表達(dá)HULC后,Akt蛋白表達(dá)水平?jīng)]有變化,Akt蛋白磷酸化水平也沒有變化,證明HULC沒有參與炎癥誘導(dǎo)的胰島素抵抗。
[Abstract]:Diabetes mellitus (DM) is an endocrine and metabolic disease, which is caused by insufficient insulin secretion, which leads to hyperglycemia and eventually to the disorder of protein and fat metabolism. At present, diabetes is a serious threat to human health, the 2016 World Health Organization survey shows that the prevalence of diabetes among adults in China is close to 10, the situation is very serious. Insulin resistance (insulin resistance,IR) is a major cause of type 2 diabetes. In individuals with insulin resistance, a normal dose of insulin does not allow fat, skeletal muscle and liver to take glucose from the blood. The sustained increase of blood glucose makes the islets secrete more insulin compensatively to maintain the stability of blood glucose, and the islet 尾 cells can not compensate for hyperglycemia, which will lead to metabolic syndrome and type 2 diabetes. This study focused on whether GBP5 and HULC were involved in insulin resistance induced by inflammatory cytokines. GBP5 belongs to the guanylate binding protein GBPs family and can be highly induced by bacterial lipopolysaccharide (LPS). In addition, GBP5 can promote the assembly and immunity of mammalian NLRP3 inflammatory bodies. HULC is a kind of lncRNA. At present, the new research on lncRNA involves a wide range of biological processes, such as development, differentiation, tissue damage, repair, regeneration, and metabolism. New disease-related lncRNA genes are also being discovered and their molecular mechanisms are being discovered and elucidated step by step. However, it is not clear whether the expression of GBP5 and HULC is regulated by inflammatory factors, and whether GBP5 and HULC regulate insulin resistance in liver is not clear. Therefore, whether inflammatory factors affect the expression of GBP5 and HULC, and whether GBP5 and HULC regulate insulin resistance induced by liver inflammation are investigated. The results are as follows: in the first part, through the common PCR and Realtime PCR tests, it was found that inflammatory factors (TNF- 偽, IFN- 緯 mixture) could promote the high expression of GBP5 after treatment with Hepa1 cells. The level of GBP5 expression in the inflammatory factor treated group was about 2200 times higher than that in the control group. According to the above results, liver cells were treated with TNF- 偽 and IFN- 緯 alone. It was found that in Hepa1 cells, the expression of GBP5 in TNF- 偽 treatment group increased about 1.7-fold, and GBP5 expression in IFN- 緯 group increased about 700fold. In hepatic parenchyma cells, the expression of GBP5 in TNF- 偽 treated group increased more than 5 times. This study demonstrated that inflammatory factors can promote the high expression of GBP5. So is GBP5 involved in inflammation-induced insulin resistance? In order to explore this problem, we constructed GBP5 overexpression adenovirus. After overexpression in primary liver parenchymal cells, we detected the insulin induced Akt phosphorylation level, and then determined whether GBP5 is involved in inflammatory insulin resistance. The results showed that the expression of Akt protein and the phosphorylation of Akt protein decreased after overexpression of GBP5, which suggested that overexpression of GBP5 might be involved in insulin resistance induced by inflammation. The second part: through the ordinary PCR and Realtime PCR validation, it was found that TNF- 偽 could promote the high expression of HULC in HepG2 cells, and the HULC expression level in the treated group was about 2.5 times higher than that in the serum-free group. In order to further investigate whether HULC is involved in insulin resistance induced by inflammation, we constructed HULC overexpression adenovirus. After overexpression in HepG2 cells, the insulin induced Akt phosphorylation level was detected. There was no change in Akt protein expression and no change in Akt phosphorylation level, which suggested that HULC was not involved in insulin resistance induced by inflammation.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
本文編號：2374038
[Abstract]:Diabetes mellitus (DM) is an endocrine and metabolic disease, which is caused by insufficient insulin secretion, which leads to hyperglycemia and eventually to the disorder of protein and fat metabolism. At present, diabetes is a serious threat to human health, the 2016 World Health Organization survey shows that the prevalence of diabetes among adults in China is close to 10, the situation is very serious. Insulin resistance (insulin resistance,IR) is a major cause of type 2 diabetes. In individuals with insulin resistance, a normal dose of insulin does not allow fat, skeletal muscle and liver to take glucose from the blood. The sustained increase of blood glucose makes the islets secrete more insulin compensatively to maintain the stability of blood glucose, and the islet 尾 cells can not compensate for hyperglycemia, which will lead to metabolic syndrome and type 2 diabetes. This study focused on whether GBP5 and HULC were involved in insulin resistance induced by inflammatory cytokines. GBP5 belongs to the guanylate binding protein GBPs family and can be highly induced by bacterial lipopolysaccharide (LPS). In addition, GBP5 can promote the assembly and immunity of mammalian NLRP3 inflammatory bodies. HULC is a kind of lncRNA. At present, the new research on lncRNA involves a wide range of biological processes, such as development, differentiation, tissue damage, repair, regeneration, and metabolism. New disease-related lncRNA genes are also being discovered and their molecular mechanisms are being discovered and elucidated step by step. However, it is not clear whether the expression of GBP5 and HULC is regulated by inflammatory factors, and whether GBP5 and HULC regulate insulin resistance in liver is not clear. Therefore, whether inflammatory factors affect the expression of GBP5 and HULC, and whether GBP5 and HULC regulate insulin resistance induced by liver inflammation are investigated. The results are as follows: in the first part, through the common PCR and Realtime PCR tests, it was found that inflammatory factors (TNF- 偽, IFN- 緯 mixture) could promote the high expression of GBP5 after treatment with Hepa1 cells. The level of GBP5 expression in the inflammatory factor treated group was about 2200 times higher than that in the control group. According to the above results, liver cells were treated with TNF- 偽 and IFN- 緯 alone. It was found that in Hepa1 cells, the expression of GBP5 in TNF- 偽 treatment group increased about 1.7-fold, and GBP5 expression in IFN- 緯 group increased about 700fold. In hepatic parenchyma cells, the expression of GBP5 in TNF- 偽 treated group increased more than 5 times. This study demonstrated that inflammatory factors can promote the high expression of GBP5. So is GBP5 involved in inflammation-induced insulin resistance? In order to explore this problem, we constructed GBP5 overexpression adenovirus. After overexpression in primary liver parenchymal cells, we detected the insulin induced Akt phosphorylation level, and then determined whether GBP5 is involved in inflammatory insulin resistance. The results showed that the expression of Akt protein and the phosphorylation of Akt protein decreased after overexpression of GBP5, which suggested that overexpression of GBP5 might be involved in insulin resistance induced by inflammation. The second part: through the ordinary PCR and Realtime PCR validation, it was found that TNF- 偽 could promote the high expression of HULC in HepG2 cells, and the HULC expression level in the treated group was about 2.5 times higher than that in the serum-free group. In order to further investigate whether HULC is involved in insulin resistance induced by inflammation, we constructed HULC overexpression adenovirus. After overexpression in HepG2 cells, the insulin induced Akt phosphorylation level was detected. There was no change in Akt protein expression and no change in Akt phosphorylation level, which suggested that HULC was not involved in insulin resistance induced by inflammation.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
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