炎性因子TNF-α和IL-1β对胰岛β细胞TXNIP表达的影响及其可能机制

发布时间：2018-05-10 15:33

本文选题：TNF-α + IL-1β　；参考：《山西医科大学》2017年硕士论文

【摘要】：研究背景:糖尿病(Diabetes Mellitus,DM)是一种由遗传和环境因素共同作用而引起的以慢性高血糖为特征的代谢性疾病,其本质是胰岛素的分泌和(或)作用缺陷。当前全世界范围内糖尿病的发病率和患病率都呈明显上升趋势,而我国作为一个人口庞大的发展中国家,已成为世界第一糖尿病大国。糖尿病主要分为1型和2型,但不论是哪种类型的糖尿病,胰岛β细胞损伤和凋亡都是糖尿病发生发展的重要原因。TNF-α和IL-1β是糖尿病时存在的慢性系统性炎症的重要介导因子,可以诱导胰岛细胞凋亡,因而在糖尿病中发挥着重要作用。硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)是目前发现的唯一内源性硫氧还蛋白(thioredoxin,TRX)结合及抑制蛋白,可以削弱TRX的抗自由基和抗凋亡功能。糖尿病时各组织TXNIP表达明显增加,研究发现高糖可以诱导INS-1细胞TXNIP m RNA和蛋白水平显著升高且伴有明显细胞凋亡。本实验室前期研究证实过表达TXNIP可通过caspase-8、9及PERK参与的内质网应激途径诱导INS-1细胞凋亡,因此,TXNIP被认为是连接糖尿病和β细胞死亡的关键环节。那么,炎性因子TNF-α和IL-1β是否可以影响INS-1细胞TXNIP的表达进而影响凋亡,若可其机制又如何?本研究的目的就在于探讨TNF-α和IL-1β诱导TXNIP变化情况及其可能机制,从而为糖尿病的预防和治疗提供新的干预靶点。目的:在正常糖脂浓度下培养INS-1细胞,外源性给予TNF-α(5ng/ml)和IL-1β(10ng/ml)刺激细胞24h,观察TNF-α和IL-1β是否引起INS-1细胞TXNIP表达变化并研究其可能机制。方法:1.实验分组:将正常糖脂浓度下培养的INS-1细胞分四组,分别为正常对照组、TNF-α组(5ng/ml,24h)、IL-1β组(10ng/ml,24h)和TNF-α+IL-1β组(5ng/ml+10ng/ml,24h)。2.外源性给予炎性因子TNF-α和IL-1β干预INS-1细胞,用CCK-8检测细胞存活率。3.流式细胞术和cleaved caspase-3两种方法检测TNF-α和IL-1β作用下INS-1细胞凋亡情况。4.Real-time PCR法检测TNF-α和IL-1β作用下INS-1细胞中TXNIP、NFκB和Ch REBP m RNA的水平。5.Western blot法检测TNF-α和IL-1β作用下INS-1细胞中TXNIP、NFκB、Ch REBP和FOXO1的蛋白表达量。6.Real-time PCR法和Western blot法检测NFκB抑制剂PDTC、p38MAPK抑制剂SB203580预处理后,INS-1细胞TXNIP、NFκB和Ch REBP m RNA和蛋白表达量。结果:1.TNF-α和IL-1β降低INS-1细胞的存活率使用不同浓度炎性因子干预INS-1细胞后,与正常对照组相比,TNF-α和IL-1β都可以浓度依赖性地诱导INS-1细胞损伤,降低细胞的存活率;且TNF-α在(5ng/ml,24h)与IL-1β在(10ng/ml,24h)时引起较明显的细胞活力下降,因此设定后期实验的TNF-α和IL-1β作用浓度分别为5ng/ml和10ng/ml,作用时间为24h。2.TNF-α和IL-1β诱导INS-1细胞凋亡Annexin-V染色流式细胞仪分析的结果显示,与对照组相比,TNF-α组、IL-1β组和TNF-α+IL-1β组细胞的凋亡率都显著升高(P0.05),Western blot结果中代表凋亡的cleaved caspase-3表达量也升高明显,提示炎性因子TNF-α和IL-1β可以引起INS-1细胞凋亡的增加,当二者联合作用时发挥协同作用,诱导更加显著的细胞凋亡。3.TNF-α和IL-1β诱导TXNIP m RNA表达上调与对照组相比,TXNIP m RNA表达水平在5ng/ml的TNF-α和10ng/ml的IL-1β时达到高峰;而选用不同作用时间的实验也表明,此浓度下作用24h时TXNIP m RNA表达水平也显著升高。因此,后续实验分组为正常对照组、TNF-α组(5ng/ml,24h)、IL-1β组(10ng/ml,24h)和TNF-α+IL-1β组(5ng/ml+10ng/ml,24h)。Real-time PCR结果显示,各炎性因子干预组,包括TNF-α+IL-1β共同作用组,TXNIP m RNA较对照组有显著升高(P0.05)。4.TNF-α和IL-1β诱导TXNIP蛋白表达量升高与对照组相比,TNF-α组、IL-1β组和TNF-α+IL-1β组细胞TXNIP蛋白水平升高明显(P0.05)。5.p-NFκB、Ch REBP、FOXO1等转录因子的测定5.1炎性因子上调Ch REBP m RNA和蛋白水平与对照组相比,TNF-α、IL-1β和二者联合作用,引起细胞Ch REBP m RNA和蛋白水平都显著升高(P0.05)。5.2炎性因子下调FOXO1蛋白表达接受TNF-α和IL-1β刺激后,各组细胞FOXO1蛋白表达量与对照组相比都显著下降(P0.05)。5.3炎性因子上调NFκB表达与对照组相比,TNF-α和IL-1β作用下细胞NFκB的m RNA水平显著升高,同时蛋白检测显示p-NFκB水平也上升(P0.05)。5.4 NFκB抑制剂PDTC下调炎性因子诱导的TXNIP和Ch REBP的表达应用20μM PDTC预处理INS-1细胞6h后,与炎性因子共同孵育细胞,PDTC显著抑制炎性因子TNF-α、IL-1β诱导的TXNIP、Ch REBP的m RNA和蛋白表达(P0.05),提示炎性因子可能通过调节NFκB和Ch REBP来增加TXNIP表达,且NFκB作为上游转录因子参与调控Ch REBP。5.5 p38MAPK抑制剂SB203580减弱炎性因子诱导的p-NFκB的激活和TXNIP的表达应用20μM SB203580预处理INS-1细胞6h后,发现炎性因子作用下INS-1细胞磷酸化NFκB和TXNIP表达量都显著降低(P0.05),提示p38MAPK作为炎症反应中的一种重要蛋白,可以通过激活核转录因子κB来诱导TXNIP改变。结论:1.炎性因子TNF-α、IL-1β可以诱导INS-1细胞凋亡。2.炎性因子TNF-α、IL-1β诱导TXNIP表达与INS-1细胞凋亡有相关性。3.炎性因子TNF-α、IL-1β可能通过p38MAPK-NFκB-Ch REBP通路上调TXNIP的表达。
[Abstract]:Research background: Diabetes Mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia caused by the combination of genetic and environmental factors. Its essence is insulin secretion and (or) deficiency. The prevalence and prevalence of diabetes in the world are obviously rising, and China is a one. The large population of developing countries has become the world's largest diabetes country. Diabetes is mainly divided into type 1 and type 2. However, no matter which type of diabetes, islet beta cell damage and apoptosis are important causes for the development of diabetes,.TNF- A and IL-1 beta are important mediating factors for chronic systemic inflammation in diabetes. In order to induce islet cell apoptosis, it plays an important role in diabetes. Thioredoxin interacting protein (TXNIP) is the only endogenous thioredoxin (thioredoxin, TRX) binding and inhibitory protein found at present. It can reduce the anti free radical and anti apoptosis function of TRX. All groups in diabetes mellitus The expression of TXNIP was significantly increased. The study found that high sugar could induce a significant increase in the level of TXNIP m RNA and protein in INS-1 cells with obvious apoptosis. This laboratory study confirmed that the expression of TXNIP could induce the apoptosis of INS-1 cells through the endoplasmic reticulum stress pathway involved in caspase-8,9 and PERK. Therefore, TXNIP is considered to be linked to diabetes. The key link of beta cell death. Then, whether the inflammatory factors TNF- alpha and IL-1 beta can affect the expression of TXNIP in INS-1 cells and then affect the apoptosis, and what is the mechanism? The aim of this study is to explore the possible mechanisms of TNF- and IL-1 beta induced TXNIP changes and the possible mechanisms to provide new intervention for the prevention and treatment of diabetes. Target. Objective: to cultivate INS-1 cells under normal sugar and fat concentration, and to give exogenous TNF- alpha (5ng/ml) and IL-1 beta (10ng/ml) to stimulate cell 24h, and observe whether TNF- A and IL-1 beta cause INS-1 cell TXNIP expression changes and study its possible mechanism. Methods: 1. experimental groups: four groups of INS-1 cells cultured under normal glycolipid concentration were divided into normal pairs, respectively. Group TNF- alpha (5ng/ml, 24h), IL-1 beta group (10ng/ml, 24h) and TNF- alpha +IL-1 beta group (5ng/ml+10ng/ml, 24h) were given inflammatory factors TNF- alpha and beta interfering cells. The cell survival rate was detected by flow cytometry and two methods for detecting apoptosis of the cells under the action of alpha and beta. CR method was used to detect TXNIP, NF kappa B and Ch REBP m under the action of TNF- alpha and IL-1 beta. TXNIP, NF kappa B and Ch REBP m RNA and protein expression. Results: the survival rate of 1.TNF- A and IL-1 beta INS-1 cells was reduced by the interference of different concentrations of inflammatory factors in INS-1 cells. 1 beta caused a significant decrease in cell viability at (10ng/ml, 24h). Therefore, the concentration of TNF- alpha and IL-1 beta in the later experiment was 5ng/ml and 10ng/ml respectively. The effect time was 24h.2.TNF- A and IL-1 beta induced INS-1 cell apoptosis by Annexin-V staining flow cytometry. The apoptosis rate of L-1 beta group increased significantly (P0.05), and the expression of cleaved caspase-3 representing apoptosis was also increased in Western blot, suggesting that the inflammatory factor TNF- alpha and IL-1 beta could induce the increase of INS-1 cell apoptosis. The synergistic effect was played when the two combined action, inducing more significant apoptosis.3.TNF- a and IL-1 beta induction. The expression level of TXNIP m RNA was higher than that of the control group, and the expression level of TXNIP m RNA reached the peak at 5ng/ml TNF- A and 10ng/ml IL-1 beta. The results of group (10ng/ml, 24h) and TNF- alpha +IL-1 beta group (5ng/ml+10ng/ml, 24h).Real-time PCR showed that every inflammatory factor intervention group, including TNF- alpha +IL-1 beta group, was significantly higher than the control group. The level of cell TXNIP protein increased significantly (P0.05).5.p-NF kappa B, Ch REBP, FOXO1 and other transcription factors. The 5.1 inflammatory factors up regulation of Ch REBP m RNA and protein levels compared with the control group. After F- alpha and IL-1 beta stimulation, the expression of FOXO1 protein in each group decreased significantly compared with the control group (P0.05).5.3 inflammatory factor up regulation of NF kappa B expression compared with the control group. The NF kappa B M levels increased significantly under the action of TNF- A and IL-1 beta. The expression of factor induced TXNIP and Ch REBP used 20 mu M PDTC to pretreat INS-1 cell 6h and incubate cells with inflammatory factors together. PDTC significantly inhibits the inflammatory factor TNF- alpha, IL-1 beta induced TXNIP. Transcriptional factors involved in the regulation of the activation of p-NF kappa B induced by Ch REBP.5.5 p38MAPK inhibitor SB203580 and the expression of TXNIP in 20 u M SB203580 preprocessing INS-1 cell 6h. An important protein can induce TXNIP change by activating nuclear factor kappa B. Conclusion: 1. inflammatory factor TNF- alpha, IL-1 beta can induce the.2. inflammatory factor TNF- alpha in INS-1 cells. IL-1 beta induced TXNIP expression and INS-1 cell apoptosis are related to.3. inflammatory factor TNF- alpha. Expression.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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