【作者】 陈洁；

【导师】 陈莹莹； 沈岳良；

【作者基本信息】 浙江大学 ， 生理学， 2012， 硕士

【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(P<0.01)；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(P<0.01)。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(P<0.05)。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(P<0.01)。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。更多还原

【Abstract】 BackgroundDiabetes mellitus has long been recognized as an independent risk factor for several forms of cardiovascular disease in both men and women. Indeed, cardiovascular complications are now the leading causes of illness and death in the diabetic patients. Many studies provide direct evidence for a contribution of inflammatory responses to the pathogenesis of atherosclerosis in type2diabetes mellitus. Although the association of diabetes mellitus-induced inflammation has been evident for several years, the triggering factor which will initiate the early cascade of inflammation is still unknown. Recently, bacterial lipopolysaccharide (LPS) has been reported to be responsible for the onset of diabetes mellitus as an important molecular origin of low-grade inflammation. The gastrointestinal tract is the biggest bacteria and LPS source of human body. Since the intestinal epithelial cells provide a physical and function barrier to prevent bacterial and LPS translocation, the circulating LPS level is very low in health person. So we hypothesized that the diabetes mellitus induced increase in LPS might be due to the intestinal epithelial barrier dysfunction. Meanwhile, the inflammatory response to the LPS stimulus is also increased in diabetic patients, in comparison to non-diabetic individuals. Since the vascular endothelium contributes to inflammatory responses in the pathogenesis of cardiovascular disease, we hypothesized that the vascular endothelium might more sensitive to the LPS stimulus in diabetic patients.Objectives1. To investigate that whether changes in proliferation of intestinal stem cell could be responsible for increased LPS in response to a high-fat diet.2. To investigate the effect and mechanism of high glucose on the exacerbation of an inflammatory response in endothelial cells.Methods1. Four-week-old male C57BL/6J mice were fed a high-fat diet (containing40%fat) for8weeks to establish insulin resistant model. Serum endotoxin level was detected using a mouse lipopolysaccharides ELISA Kit. Insulin resistance was calculated according to the homeostasis model assessment (HOMA) calculation: fasting glucose (mmol/L)×fasting insulin (mU/L)/22.5. The xylose absorption assay was performed to evaluate the intestinal barrier function. Epithelial cell proliferation was evaluated by assessing BrdU incorporation.2. Human umbilical vein endothelial cells (HUVECs) were exposed to different concentration of glucose (5.5,25, or50mmol/L) or/and LPS (0,10,100, or1000μg/L) for24h. Levels of IL-6and TNF-a were detected by ELISA assay. The expression of TLR2/4and NF-kB was evaluated by western blot.Results1. Compared with control group, high fat diet caused the increase in insulin resistance index after8w of diet. A40%high fat diet increased LPS1.5-fold when compared with control fed mice. The intestinal barrier function and the total number of proferating cells per crypt were declined in mice fed with high fat diet (P<0.01).2. Under high glucose conditions, LPS-induced production of TNF-a and IL-6increased in HUVECs (P＜O.05). Although25mmol/L of glucose or LPS (100μg/L) alone could not influence the expression of TLR2and NF-kB, coincubation with glucose and LPS could increase the expression of TLR2and NF-kB. LPS (100μg/L) did not alter the TLR4expression in HUVECs even under high glucose condition.Conclusion1. The data indicated that high-fat diet could increase serum LPS in mice, which might involved in decline in proliferation of intestinal stem cell and dysfunction of intestinal barrier.2. The results also suggested that augmented TNF-alpha and IL-6production induced by LPS in HUVECs under high glucose condition is due to high glucose-induced increase in NF-kappaB-dependent activation of TLR2.更多还原