GBP5和HULC在炎症诱导的胰岛素抵抗中的作用机制研究

发布时间：2018-12-12 05:49

【摘要】：糖尿病是一种内分泌代谢性疾病,其诱因是由于胰岛素分泌不足,进而导致血糖过高,最终引起蛋白质和脂肪的代谢发生紊乱。目前,糖尿病严重威胁着人类的健康,2016年的世界卫生组织调查显示,目前我国成年人的糖尿病发病率已经接近10%,形势十分严峻。普遍研究表明,胰岛素抵抗(insulin resistance,IR)是导致2型糖尿病的主要原因,在胰岛素抵抗个体中,正常量的胰岛素不能使脂肪、骨骼肌和肝脏从血液中摄取葡萄糖,血糖持续升高,使胰岛代偿性分泌更多的胰岛素以维持血糖的稳定,而胰岛β细胞不能代偿高血糖将导致代谢综合征和2型糖尿病。本课题主要针对GBP5和HULC是否参与炎症因子诱导的胰岛素抵抗进行研究。GBP5属于鸟苷酸结合蛋白GBPs家族的一种,可以被细菌脂多糖(LPS)高诱导。另外,GBP5还能够促进哺乳动物NLRP3炎症小体的组装和免疫。而HULC属于lncRNA的一种,目前对于lncRNA的新兴的研究涉及到发育,分化,生长组织损伤、修复、再生,及代谢的广泛的生物过程。新的疾病相关lncRNA基因也正在发现当中,并且其分子机制正在被一步一步发现和阐明。但是,GBP5和HULC的表达调控是否受炎症因子调控还不清楚,并且,GBP5和HULC是否调节肝脏胰岛素抵抗也还不清楚。为此,本课题针对炎症因子是否影响GBP5和HULC的表达水平,以及GBP5和HULC是否调节肝脏炎症诱导的胰岛素抵抗进行了探究。研究结果如下:第一部分:通过普通PCR验证和Realtime PCR验证,发现炎症因子(TNF-α、IFN-γ混合物)处理Hepa1细胞后,可以促进GBP5高表达,且炎症因子处理组比对照组GBP5表达水平高2200倍左右。根据以上实验结果,采用TNF-α和IFN-γ单独处理肝脏细胞,发现在Hepa1细胞中,TNF-α处理组GBP5的表达升高了1.7倍左右,IFN-γ处理组GBP5表达升高了700倍左右;而在肝实质细胞中,TNF-α处理组GBP5的表达升高了5倍以上。此实验证明炎症因子可以促进GBP5的高表达。那么GBP5是否参与了炎症诱导的胰岛素抵抗呢?为了探究这个问题,本课题构建了GBP5过表达腺病毒,在原代肝实质细胞中过表达后,检测胰岛素诱导的Akt磷酸化水平,进而判断GBP5是否参与炎症诱导的胰岛素抵抗。结果发现过表达GBP5后,Akt蛋白表达水平降低,Akt蛋白磷酸化水平降低,证明过表达GBP5可以参与炎症诱导的胰岛素抵抗。第二部分:通过普通PCR验证和Realtime PCR验证,发现炎症因子TNF-α处理HepG2细胞后,可以促进HULC高表达,且炎症因子处理组比无血清处理组的HULC表达水平高2.5倍左右。为了进一步探究HULC是否参与了炎症诱导的胰岛素抵抗,本课题构建了HULC过表达腺病毒,在HepG2细胞中过表达后,检测胰岛素诱导的Akt磷酸化水平,结果发现过表达HULC后,Akt蛋白表达水平没有变化,Akt蛋白磷酸化水平也没有变化,证明HULC没有参与炎症诱导的胰岛素抵抗。
[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- 伪, IFN- 纬 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- 伪 and IFN- 纬 alone. It was found that in Hepa1 cells, the expression of GBP5 in TNF- 伪 treatment group increased about 1.7-fold, and GBP5 expression in IFN- 纬 group increased about 700fold. In hepatic parenchyma cells, the expression of GBP5 in TNF- 伪 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- 伪 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.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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