【作者】 李桂林；

【导师】 梁尚栋；

【作者基本信息】 南昌大学 ， 外科学， 2011， 博士

【摘要】 研究背景：糖尿病是危害人类健康的四大疾病之,目前我国是世界上糖尿病发病人数最多的国家。糖尿病严重影响着人民健康和社会发展,对糖尿病的防治已成为医学科学工作者的一个重要课题。糖尿病的主要危害不是糖尿病本身,而是在糖尿病发展过程中出现的各种慢性并发症。糖尿病引发的交感神经和心血管损伤并发症严重影响患者的生活质量,是致残、致死的重要原因。血管内皮细胞是具有血管调节功能和分泌作用的重要器官。颈上交感神经节的节后神经纤维可分布至心神经丛,影响心脏和血管的活动。研究报道莲籽芯具有降血糖作用。甲基莲心碱(neferine, Nef)是从睡莲科植物莲成熟种子的绿色胚芽(莲籽芯)中提取的一种双苄基异喹啉类生物碱。本实验研究探索甲基莲心碱在糖尿病及其并发交感神经和心血管损伤中的作用及其可能作用的新靶点,并为糖尿病及其并发症的防治提供新的实验依据。目的：1、通过观察高糖致血管内皮细胞基因表达变化及甲基莲心碱的影响,了解甲基莲心碱对高糖致血管内皮细胞损伤的保护作用及可能机理；2、几乎全部与人类疾病有关的基因与大鼠基因有同源性。实验观察甲基莲心碱对2型糖尿病模型大鼠血糖、血脂、血胰岛素及血压影响的同时,了解甲莲心碱对糖尿病模型大鼠颈上交感神经节CCL5及CCR5表达的影响，探讨糖尿病及其并发症的发病机制及甲基莲心碱的作用机理；3、应用差异蛋白质组学方法观察糖尿病模型大鼠颈上交感神经节相关蛋白质分子的变化,筛选药效作用靶点,揭示甲基莲心碱防治糖尿病并发症的作用机理,为临床推广应用提供实验依据。方法：1.以高浓度(44mmM)的葡萄糖作用于原代培养的人脐静脉内皮细胞(HUVEC)共孵育5天诱导模拟糖尿病内皮细胞损伤。应用硝酸还原酶法检测细胞上清液中一氧化氮(NO)的含量；荧光法测定细胞内活性氧(ROS)水平；美国Superarray RT2 ProfilerTM PCR基因表达谱芯片进行多基因分析；通过免疫组织化学、原位杂交、RT-PCR和蛋白印迹分析从内皮细胞基因表达谱中筛选出的和糖尿病并发症有重要相关性的趋化因子5(CCL5)及其受体CCR5mRNA和蛋白表达变化,并观察甲基莲心碱对其影响。2.选用雄性SD大鼠,通过高糖高脂饮食加腹腔小剂量注射链脉佐菌素复制糖尿病大鼠模型,运用生化、放免、血压测定、双向电泳、免疫组织化学、原位杂交、RT-PCR和蛋白印迹等检测手段和方法,观察甲基莲心碱对糖尿病大鼠体重、空腹血糖、血脂、血胰岛索、血压及颈上神经节和心肌组织CCL5及其受体CCR5mRNA和蛋白表达的影响。3.通过差异蛋白质组学技术筛选出糖尿病大鼠模型颈上交感神经节相关蛋白质的变化并观察甲基莲心碱的作用,并将其中与糖尿病并发症有重要相关性的二甲基精氨酸氨基水解酶1(DDAH1)和泛素羧端水解酶L1 (UCHL1)蛋白进一步应用蛋白印迹方法检测,确定甲基莲心碱对神经节DDAH1和UCHL1表达的影响。结果：1、高糖孵育HUVEC细胞5天后,细胞培养上清液中NO含量显著性减少,细胞内ROS水平明显升高；高糖同时使用甲基莲心碱能显著性增加高糖所致NO含量的减少,降低高糖所致ROS水平的升高。PCR芯片结果发现慢性高糖处理后有16个基因上调,10个基因下调,其中变化最大的是与内皮细胞活性和内皮细胞损伤有关的CCL5。RT-PCR证实高糖培养细胞CCL5和CCR5mRNA增加,甲基莲心碱能纠正高糖引起的CCL5和CCR5 mRNA的表达上调。蛋白印迹结果发现慢性高糖引起CCL5和CCR5蛋白表达上调,甲基莲心碱能显著降低高糖所致的CCL5和CCR5蛋白表达的上调。2、甲基莲心碱处理后使2型糖尿病模型大鼠体重增加,血压降低,心率减慢,空腹血糖、血胰岛素、总胆固醇、甘油三酯降低,高密度脂蛋白升高,即甲基莲心碱具有较好的改善糖尿病大鼠胰岛素抵抗和调整脂代谢紊乱作用。RT-PCR和原位杂交结果显示糖尿病大鼠颈上神经节和心肌组织中CCL5和CCR5 mRNA的表达上调,甲基莲心碱能降低CCL5和CCR5mRNA的表达上调。免疫组织化学和蛋白印迹结果发现糖尿病大鼠颈上神经节和心肌组织中CCL5和CCR5蛋白的表达增加,甲基莲心碱可降低CCL5和CCR5蛋白的增加表达。3、差异蛋白质组学技术分离、鉴定糖尿病大鼠颈上神经节差异蛋白结果显示：差异表达的23个蛋白质斑点经鉴定后,归为17种蛋白,这17种蛋白按功能分为细胞骨架、能量代谢、信号传导、NO生成调节、氧化应激和炎症反应六大类。糖尿病模型大鼠颈上交感神经节大部分蛋白表达量均不同程度下调,只有NADH脱氢酶表达量上调。甲基莲心碱能纠正糖尿病大鼠大部分改变的蛋白表达量。二维电泳及蛋白印迹检测结果证实糖尿病大鼠颈上神经节和心肌组织DDAH1和UCHL1表达降低。DDAH1参与NO生成的调节,UCHL1与氧化应激有关。甲基莲心碱可增加DDAH1和UCHL1蛋白表达。结论：高糖可明显影响血管内皮细胞和颈上交感神经节mRNA和蛋白表达。甲莲心碱可以降低糖尿病内皮细胞、2型糖尿病模型大鼠颈上神经节和心肌中CCL5和CCR5的表达,增加糖尿病大鼠颈上神经节和心肌组织DDAH1和UCHL1表达,从而增加NO的生成,减少ROS的产生,对糖尿病及其血管内皮细胞和交感神经损伤产生防治作用。更多还原

【Abstract】 Introduction:Diabetes is one of four desease threatened to human health. The incidence of diabetic disease in China is highest in the world. Diabetes is seriously harm to human health and the social development. Prevention and herapy to diabetes have been thought as an important task for medical researchers. The main harm for diabetic is the chronic complicantions. The complicantions of sympathetic nerve and cardiovascular injury induced by diabetes are the most popular and seriously threatening factors to the living quality of diabetic patients. The complicantions of diabetic also are the major causes of disablity and death. Vascular endothelial cells (VEC) as the critical organ are possessed with vascular regulation and endocrine function. The postganglionic fibers of superior cervical ganglion (SCG) are distributed to cardiac plexus and regulate the activity of heart and vessel. It is reported that the seeds of Nelumbo micifera Gaertn have hypoglycemic activity. Neferine is a bisbenzylisoquinline alkaloid isolated from a Chinese medicinal herb (Nelumbo micifera Gaertn). The aim of this study was to investigate effects of neferine on the diabetes and its associated complications of sympathetic injury and the dysfunctional vascular endothelium, then provide experimental data for prevention and herapy to diabetes.Objective:1. The aim of the studies was to investigate the protective effect and its’mechanism of neferine against damage of VEC induced by chronic high glucose (HG) by ovserving the change of gene expression in VEC induced by HG and the effect of nefferin on the change of gene expression.2. Almost all human genes known to be associated with disease have orthologues in the rat genome. The studies observed the effects of neferine on the blood glucose, blood lipid, blood insuline, blood pressure of model rat for 2 type diabeteic and the expressions of CCL5 and CCR5 in SCG of diabetic rats. The mechanisms of neferine acting to the pathogenesy of diabetes and its complications were investigated.3. The change of relavant protein in SCG of diabetic rats was investigated by differential-disply proteomics. Drug target to reveal the effective mechamism of neferine in prevention and cure diabetic complications was screened. The studies provide experimental basis for clinical application.Methods:1. The primary cultured human umbilical vein endothelial cells (HUVECs) were treated with glucose (44 mM) for 5 d to simulate the model of endothelial cells injury. The content of nitric oxide (NO) in the cultured medium was measured by nitrate reductase method. The level of intracellular reactive oxygen species (ROS) was detected by fluorospectrophotometer analysis. Multiple genes analysis was undertaken by Superarray RT2 ProfilerTM PCR. array. The expressions of CCL5 and CCR5 screened from genetic expression and associated with diabetic complications were detected by immunohistochemistry, in situ hybridization, RT-PCR and western blot.2. Diabetic model of SD male rat was established with high calorie diet and intrapcritoneal injection of low dose streptozotocin. Methods of multiple studies were used as including biochemistry, radiommunoassay. two-dimensional electrophoresis, immunohistochemistry, in situ hybridization, RT-PCR and western blot. The studies were used to observe the effect of neferine on diabetic rat body weight, fasting blood glucose, insulin, lipid, blood pressure, the expression of CCL5 and CCR5 mRNA and protein in SCG and myocardium.3. The change of relavant protein in SCG of diabetic rats was identified by using differential-disply proteomics. The studies reveal the effect of neferine on diabetic complications. The studies provide experimental basis for clinical application. The expression of the dimethylarginine dimethylaminohydrolase 1 (DDAH1) and ubiquitin carboxy terminal hydrolase L1(UCHL1) in SCG was tesed by western blot. Effect of of neferine on the expression of the proteins (DDAH1 and UCHL1) associated with diabetic complications was confirmed..Results:1. Compared with control group, the content of NO in the medium was significantly decreased when exposure of HUVECs to high glucose for 5 days and the level of intracellular ROS was increased. Neferine significantly enhanced the decreased level of NO and decreased the increased level of intracellular ROS. The result of PCR Array showed that there were 16 genes upregulation and 10 genes downregulation in HUVECs following chronic culture with high glucose. Of these, CCL5 was the most significantly upregulation. The expressions of CCL5 and CCR5 mRNA and protein were upregulation in HUVECs treated with high glucose. The elevated expressions of CCL5 and CCR5 mRNA and protein were inhibited by neferine.2. Blood pressure,heart rate, fasting blood glucose, insulin, total cholesterol and triglyceride were decreased and body weight, high density lipoprotein were increased in 2 type diabetic model rats treated with neferine. The results of RT-PCR and in situ hybridization indicated that the expressions of CCL5 and CCR5 mRNA were enhanced in SCG and myocardium of diabetic rats. The expressions of CCL5 and CCR5 protein detected by immunohistochemistry and western blot were enhanced in SCG and myocardium of diabetic rats. The elevated expressions of CCL5 and CCR5 were decreased in SCG and myocardium of diabetes treated with neferine.3. The results tested by differential-disply proteomics in SCG of diabetic rats showed that 23 differential protein spots were identified and its were classified into 17 categories of proteins. Except NADH dehydrogenase was upregulation. others of proteins were downregulation in SCG of diabetic rats. The expression changes of most proteins were reverted in diabetic rats treated with neferine. Proteins identified were involved in cytoskeleton. energy metabolism, signal transduction, regulation of NO generation, oxidative stress and imflammatory reaction. DDAH1 was involved in the regulation of NO generation. UCHL1 was associated with oxidative stress. The expressions of DDAH1 and UCHL1 protein were decreased in SCG and myocardium of diabetic rats. The downregulated expressions of DDAH1 and UCHL1 protein in diabetic rats were elevated by neferine.Conclusions:High glucose can significantly affect the expressions of mRNA and protein in VEC and SCG. Neferine can decrease the upregulated expressions of CCL5 and CCR5 in endothelial cells, SCG and myocardium of diabetic rats. Neferine can also increase the downregulate expressions of DDAH1 and UCHL1 in SCG and myocardium of diabetic rats. Consequently, neferine increased the generation of NO and decreased the production of ROS. These results may suggest that neferine has preventive and therapeutic effect on endothelial cells and SCG injury induced by high glucose.更多还原