高糖对内毒素炎症反应的影响及其机制研究
发布时间：2019-03-24 11:36
【摘要】：背景 心血管病变是糖尿病的主要并发症和致死原因,而动脉粥样硬化是其心血管并发症的主要病理表现。大量的研究证实,2型糖尿病和动脉粥样硬化可能是同一病理生理基础上平行发展的两种疾病,它们的共同基础是慢性亚临床炎症。虽然已证实,糖尿病患者血液中多种炎性介质均呈明显增高趋势,但其炎症发生的真正原因还不明了。最新的研究表明,细菌来源的脂多糖(lipopolysaccharide, LPS)可能是引起糖尿病慢性亚临床炎症的真正始发因子。肠道是人体最大的细菌和LPS贮存场所。由于正常人体肠上皮细胞能作为物理及功能上的屏障以防止LPS转位,因此正常人体血液中的LPS水平非常低。所以我们推测,糖尿病血液LPS增加可能与肠上皮屏障功能减弱导致LPS转位有关。此外,糖尿病患者与健康人相比,受LPS刺激时所引发的炎症反应更加强烈。目前,已有大量证据显示糖尿病患者内皮功能异常是血管并发症的始发因素,且在心血管疾病的炎症机制中起了重要的作用。故我们推测,高糖条件下,血管内皮细胞对LPS刺激更为敏感。 目的 1.阐明高脂饮食诱导的胰岛素抵抗小鼠其血液LPS水平的升高是否与肠上皮细胞增殖能力下降引起的肠道屏障功能改变有关。 2.明确高糖能否加重LPS诱导的血管内皮细胞的炎症反应并探究其可能机制。 方-法 1.动物实验部分：用高脂饮食诱发小鼠胰岛素抵抗模型。ELISA法测定血清LPS水平。利用稳态模型评价胰岛素抵抗指数。D-木糖吸收实验测定肠黏膜通透性。免疫组织化学法观察Brdu染色阳性的细胞数,计算肠上皮细胞的增殖指数。 2.细胞实验部分：人脐静脉内皮细胞(Human umbilical vein endothelial cells, HUVECs)用不同浓度的葡萄糖(5.5,25,50mmol/L)和/或不同浓度的LPS(0,10,100,1000gg/L)孵育24h。ELISA法测定培养上清液中IL-6和TNF-a水平。Western blot去测定目标蛋白表达。 结果 1.动物实验部分：高脂饲料喂养8周后小鼠胰岛素抵抗指数明显高于正常对照组,血清LPS水平明显增高,小鼠对D-木糖吸收量明显增加(P0.01)；高脂饲料喂养8周后小鼠空肠隐窝中平均BrDu阳性细胞数减少,BrDu阳性细胞百分比下降(P0.01)。 2.细胞实验部分：与正常糖浓度组相比,细胞在高糖环境下,同样浓度的LPS引起的血管内皮细胞上清TNF-a和IL-6水平明显增高(P0.05)。虽然单纯25mmol/L高糖或100μg/L LPS孵育细胞对TLR2蛋白表达无明显影响,但两者共同孵育细胞,可诱导TLR2表达显著增加。而各组TLR4的表达却无显著变化。提示高糖和LPS对TLR2的表达有协同作用。此外,高糖和LPS共同孵育血管内皮细胞后,细胞核内NF-κB蛋白表达也显著增加(P0.01)。 结论 1.高脂饮食诱导的小鼠代谢性内毒素血症可能与肠上皮细胞的增殖修复能力下降引起的肠道屏障功能改变有关。 2.高糖可加重LPS诱导的血管内皮细胞炎症反应,其机制可能与高血糖诱导的NF-κB依赖性TLR2表达增加有关。
[Abstract]:background Cardiovascular disease is the main complication and cause of diabetes, and atherosclerosis is the main pathological form of its cardiovascular complications. A large number of studies have shown that type 2 diabetes and atherosclerosis may be two types of diseases that are parallel to the same pathophysiology, and their common basis is chronic subclinical inflammation. The number of inflammatory mediators in the blood of patients with diabetes has been shown to be significantly higher, but the true cause of its inflammation is unknown Recent studies have shown that lipopolysaccharides (LPS) of bacterial origin may be the true origin of the chronic subclinical inflammation of diabetes. The intestinal tract is the largest bacterial and LPS storage site in the human body The normal human intestinal epithelial cells can be used as a physical and functional barrier to prevent the translocation of LPS, so the level of LPS in normal human blood is very high, Low. So we have speculated that the increase in the LPS of the diabetic blood may be associated with a decrease in the function of the intestinal epithelial barrier, which results in the translocation of the LPS Off. In addition, the inflammatory response induced by LPS stimulation is more intense in patients with diabetes compared to healthy controls Currently, there is a large amount of evidence that the endothelial dysfunction in the diabetic patients is an initiating factor for vascular complications and plays an important role in the inflammatory mechanism of the cardiovascular disease It is suggested that the vascular endothelial cells are more sensitive to the stimulation of LPS in the presence of high glucose. A sense of feeling. Objective:1. To clarify whether the insulin resistance induced by high-fat diet is related to the intestinal barrier work caused by the decrease in the level of the blood LPS and the decrease of the proliferation ability of the intestinal epithelial cells. 2. clarify whether high glucose can aggravate the inflammatory response of LPS-induced vascular endothelial cells and explore Possible mechanism.-method 1. Animal experiment part: high-fat diet induced mouse insulin resistance model. ELIS A method for determination of serum LPS level. Steady state model evaluation of insulin resistance index. D-xylose absorption The number of cells positive for Brdu staining was observed by immunocytochemical method, and the number of cells positive for Brdu staining was measured by immunohistochemistry. Cell experiment: Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of glucose (5.5,25,50 mmol/ L) and/ or different concentrations of LPS (0,10,100,1000 mmol/ L) for 24 h. Serum IL-6 and TNF-a levels. Bl Results 1. The experimental part of the animal: The insulin resistance index of the mice was significantly higher than that of the normal control group after 8 weeks of high-fat feed, and the level of the serum LPS increased significantly. -The uptake of xylose was significantly increased (P0.01); the mean number of BrDu-positive cells in the crypt of the jejunum of mice after 8 weeks of high-fat diet was reduced, and BrD The percentage of u-positive cells decreased (P0.01). A and IL-6 levels were significantly higher (P0.05). Although the expression of TLR2 protein was not significantly affected by 25 mmol/ L high glucose or 100. m u.g/ L of LPS, both of them Compared with the incubation cells, the expression of TLR2 can be induced to be significant There was no significant change in the expression of TLR4 in each group. High glucose and LPS have a synergistic effect on the expression of TLR2. In addition, after incubation of the vascular endothelial cells by high glucose and LPS, NF -I'm sorry. Conclusion 1. The metabolic endotoxemia induced by high-fat diet may be closely related to the intestinal epithelium. 2. High glucose can aggravate the inflammatory response of LPS-induced vascular endothelial cells, and its mechanism may
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2446296
[Abstract]:background Cardiovascular disease is the main complication and cause of diabetes, and atherosclerosis is the main pathological form of its cardiovascular complications. A large number of studies have shown that type 2 diabetes and atherosclerosis may be two types of diseases that are parallel to the same pathophysiology, and their common basis is chronic subclinical inflammation. The number of inflammatory mediators in the blood of patients with diabetes has been shown to be significantly higher, but the true cause of its inflammation is unknown Recent studies have shown that lipopolysaccharides (LPS) of bacterial origin may be the true origin of the chronic subclinical inflammation of diabetes. The intestinal tract is the largest bacterial and LPS storage site in the human body The normal human intestinal epithelial cells can be used as a physical and functional barrier to prevent the translocation of LPS, so the level of LPS in normal human blood is very high, Low. So we have speculated that the increase in the LPS of the diabetic blood may be associated with a decrease in the function of the intestinal epithelial barrier, which results in the translocation of the LPS Off. In addition, the inflammatory response induced by LPS stimulation is more intense in patients with diabetes compared to healthy controls Currently, there is a large amount of evidence that the endothelial dysfunction in the diabetic patients is an initiating factor for vascular complications and plays an important role in the inflammatory mechanism of the cardiovascular disease It is suggested that the vascular endothelial cells are more sensitive to the stimulation of LPS in the presence of high glucose. A sense of feeling. Objective:1. To clarify whether the insulin resistance induced by high-fat diet is related to the intestinal barrier work caused by the decrease in the level of the blood LPS and the decrease of the proliferation ability of the intestinal epithelial cells. 2. clarify whether high glucose can aggravate the inflammatory response of LPS-induced vascular endothelial cells and explore Possible mechanism.-method 1. Animal experiment part: high-fat diet induced mouse insulin resistance model. ELIS A method for determination of serum LPS level. Steady state model evaluation of insulin resistance index. D-xylose absorption The number of cells positive for Brdu staining was observed by immunocytochemical method, and the number of cells positive for Brdu staining was measured by immunohistochemistry. Cell experiment: Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of glucose (5.5,25,50 mmol/ L) and/ or different concentrations of LPS (0,10,100,1000 mmol/ L) for 24 h. Serum IL-6 and TNF-a levels. Bl Results 1. The experimental part of the animal: The insulin resistance index of the mice was significantly higher than that of the normal control group after 8 weeks of high-fat feed, and the level of the serum LPS increased significantly. -The uptake of xylose was significantly increased (P0.01); the mean number of BrDu-positive cells in the crypt of the jejunum of mice after 8 weeks of high-fat diet was reduced, and BrD The percentage of u-positive cells decreased (P0.01). A and IL-6 levels were significantly higher (P0.05). Although the expression of TLR2 protein was not significantly affected by 25 mmol/ L high glucose or 100. m u.g/ L of LPS, both of them Compared with the incubation cells, the expression of TLR2 can be induced to be significant There was no significant change in the expression of TLR4 in each group. High glucose and LPS have a synergistic effect on the expression of TLR2. In addition, after incubation of the vascular endothelial cells by high glucose and LPS, NF -I'm sorry. Conclusion 1. The metabolic endotoxemia induced by high-fat diet may be closely related to the intestinal epithelium. 2. High glucose can aggravate the inflammatory response of LPS-induced vascular endothelial cells, and its mechanism may
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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