NLRP3炎性小体在2型糖尿病脑微血管内皮细胞中的变化及机制研究

发布时间：2018-04-22 06:07

本文选题：NLRP3炎性小体 + 2型糖尿病　；参考：《山东大学》2017年硕士论文

【摘要】：目的NLRP3炎性小体是大分子蛋白复合体,能够识别多种危险刺激信号,募集并激活含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,casepase-1),进一步促进炎症因子白介素-1β(interleukin-1β,IL-1β)和白介素-18(Interleukin-18,IL-18)的释放,是机体固有免疫的一部分。目前研究发现炎性小体在多种炎性相关疾病中发挥重要作用,如糖尿病肾病、视网膜病、自身免疫性疾病、动脉粥样硬化、再灌注损伤等。2型糖尿病引起的中枢神经系统损伤主要包括认知障碍和脑血管管疾病。以往研究表明NLRP3炎性小体与中枢神经系统疾病关系密切,但是炎性小体在2型糖尿病脑微血管的损伤中是否起作用,及作用机制尚不清楚。本实验通过观察大鼠脑组织中NLRP3的分布,及高糖处理小鼠脑微血管内皮细胞,检测细胞内相关蛋白,来探讨NLRP3炎性小体在高糖作用下的脑微血管内皮细胞中的变化及变化机制。方法(1)饲养6月龄雄性Wistar大鼠作为实验对照组,自发性2型糖尿病(goto-kakizaki,GK)大鼠作为实验组,每组各5只,采用免疫组织化学方法观察脑组织中NLRP3的表达及空间分布。(2)体外培养小鼠脑微血管内皮细胞。不同葡萄糖浓度培养基模拟体内2型糖尿病内环境,活性氧(Reactive Oxygen Species,ROS)清除剂N-乙酰半胱氨酸(NAC)作为抑制剂,将细胞分为对照组(培养基糖浓度5.6mmol/L)、高糖1组(HG1组,培养基糖浓度1Ommol/L)、高糖2组(HG2组,培养基糖浓度20mmol/L)、高糖3组(HG3组,培养基糖浓度30mmol/L)及高糖+NAC组(HG +NAC组,目的蛋白表达较明显组的糖浓度组内按照1:1000预先加入NAC)。(3)采用Western blotting法检测小鼠脑微血管内皮细胞各组硫氧还蛋白交互蛋白(TXNIP)和NLRP3的表达,采用ELISA法检测各组细胞中的IL-1 β的水平;采取流式细胞术评估对照组、HG3组、HG+NAC组ROS的含量。(4)采用SPSS19.0统计学软件对实验所得数据进行分析,所得数据以x±s表示,两组间的比较应用t检验,多组间的比较应用单因素方差分析。所得P0.05为差异有统计学意义。结果(1)NLRP3主要分布于GK大鼠脑皮层及皮层下组织的神经胶质细胞和微/小血管管壁。与Wistar大鼠相比,GK大鼠的阳染强度,阳染血管数均有增加(P0.05);(2)与对照组相比,HG1组、HG2组、HG3组TXNIP、NLRP3的表达有所增加(P0.01),尤以HG3组蛋白表达增加最明显。(3)与对照组相比,HG1组、HG2组、HG3组细胞内IL-1 β水平有所增高(P0.01)。(4)与对照组相比,HG3组ROS的含量增加(P0.01);给予ROS清除剂后,与HG3组相比,HG+NAC组细胞内ROS含量下降(P0.01),TXNIP、NLRP3表达水平下降(p0.01),细胞内IL-Iβ水平下降(P0.01)。结论(1)高糖可促进NLRP3和TXNIP的蛋白表达,并随糖浓度的增加而表达增高。(2)高糖各组IL-Iβ的水平含量增加,说明NLRP3炎性体被激活。(3)高糖可导致脑微血管内皮细胞产生过多ROS。给予ROS清除剂后,高糖组细胞内的ROS含量下降,且细胞内的TXNIP和NLRP3的蛋白表达、IL-1β的水平均下降,说明高糖可以通过与ROS相关的途径活化NLRP3炎性小体。(4)据此,我们推测NLRP3炎性小体在2型糖尿病脑微血管内皮细胞中可经高糖-ROS-TXNIP途径被活化,参与2型糖尿病脑微血管的损伤。
[Abstract]:Objective NLRP3 inflammatory corpuscle is a large molecular protein complex that recognizes a variety of dangerous stimulus signals and raises and activates -1 (cysteinyl aspartate specific proteinase-1, casepase-1) containing cysteine, and further promotes the inflammatory factor of interleukin -1 beta (interleukin-1 beta, IL-1 beta) and interleukin -18 (Interleukin-18) (Interleukin-18). The release of IL-18 is part of the body's inherent immunity. Current studies have found that inflammatory corpuscles play an important role in a variety of inflammatory related diseases, such as diabetic nephropathy, retinopathy, autoimmune diseases, atherosclerosis, reperfusion injury and other central nervous system injuries, including cognitive impairment and brain. Vascular diseases. Previous studies have shown that NLRP3 inflammatory corpuscles are closely related to central nervous system diseases, but the role and mechanism of inflammatory corpuscles in the brain microvascular injury of type 2 diabetes are not clear. This experiment was conducted by observing the distribution of NLRP3 in the brain tissue of rats and the high glucose treatment of the microvascular endothelial cells in the brain of mice. The changes and changes of NLRP3 inflammatory corpuscles in the cerebral microvascular endothelial cells under the action of high glucose were investigated. Methods (1) 6 month old male Wistar rats were fed as experimental control group, and spontaneous type 2 diabetes (Goto-Kakizaki, GK) rats were used as experimental group, and 5 rats in each group were observed by immunohistochemical method. The expression and spatial distribution of NLRP3 in the tissue. (2) the mouse brain microvascular endothelial cells were cultured in vitro. Different glucose concentration medium was used to simulate the internal environment of type 2 diabetes in the body. The active oxygen (Reactive Oxygen Species, ROS) scavenger N- acetylcysteine (NAC) was used as the inhibitor, and the cells were divided into the control group (culture medium sugar concentration 5.6mmol/L) and high sugar 1. Group (group HG1, glucose concentration 1Ommol/L), 2 groups of high sugar (group HG2, culture medium sugar concentration 20mmol/L), high sugar 3 groups (HG3 group, medium sugar concentration 30mmol/L) and high glucose +NAC group (HG +NAC group, the sugar concentration group of the target protein expression group was pre added into NAC) in the sugar concentration group. (3) the mouse brain microvascular endothelial cells were detected by Western assay The expression of thioredoxin interaction protein (TXNIP) and NLRP3 and the level of IL-1 beta in the cells of each group were detected by ELISA. Flow cytometry was used to evaluate the content of ROS in the control group, HG3 group and HG+NAC group. (4) the data were analyzed with SPSS19.0 statistics software, and the data were expressed in X + s, and the two groups were compared to t check. The results of P0.05 were statistically significant. Results (1) NLRP3 was mainly distributed in the neuroglia cells and microvascular wall of subcortical tissues of GK rats. Compared with Wistar rats, the positive intensity of GK rats increased (P0.05); (2) compared with the control group, the number of positive cells increased (P0.05). The expression of TXNIP in group HG1, group HG2 and HG3 increased (P0.01), especially in HG3 histone expression. (3) the level of IL-1 beta in HG1, HG2 and HG3 groups increased (P0.01) in comparison with the control group (P0.01). (4) compared with the control group, the content of the group increased. Content decreased (P0.01), TXNIP, NLRP3 expression level decreased (P0.01), IL-I beta level decreased (P0.01). Conclusion (1) high sugar can promote the protein expression of NLRP3 and TXNIP, and increase with the increase of sugar concentration. (2) the level of IL-I beta in high sugar groups increases, indicating that NLRP3 inflammatory bodies are activated. (3) high glucose can lead to microvascular endothelium finely. The content of ROS in the cells of high glucose decreased, and the protein expression of TXNIP and NLRP3 in the cells decreased, and the level of IL-1 beta in the cells decreased, indicating that the high glucose could activate the NLRP3 inflammatory corpuscle in the ROS related pathway. (4) according to this, we speculate that the NLRP3 inflammatory corpuscle is in the cerebral microvascular endothelial cells of type 2 diabetes. It can be activated by high glucose -ROS-TXNIP pathway and participate in cerebral microvascular damage in type 2 diabetes.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R747.9

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