【作者】 陈玲；

【导师】 吕湛；

【作者基本信息】 川北医学院 ， 内科学， 2010， 硕士

【摘要】 背景和目的：细胞衰老促进机体老化和老年相关疾病如动脉粥样硬化、高血压等发生。糖尿病患者的心脏事件发生率增加,病变程度加重,但其发生机制还不明确。本文探讨体外培养条件下,不同浓度的葡萄糖和胰岛素对人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)衰老及蛋白激酶B(protein kinase B,PKB/AKT)活化的影响。以期了解葡萄糖和胰岛素对心血管的作用,并进一步研讨糖尿病致动脉粥样硬化的发病机制。方法：用含10%胎牛血清的DMEM低糖培养基行体外培养人脐静脉内皮细胞,取其对数生长期细胞用于实验。实验分组：(1)葡萄糖干预组：不同浓度的葡萄糖,包括5.6mmol/L、16.7mmol/L、33mmol/L组；(2)胰岛素组：对照组、1nmol/L、10nmol/L、100nmol/L组。各组细胞培养至对数生长期,分别给与相应浓度的处理因素,作用24h,用β半乳糖酶染色法检测细胞衰老率,并用免疫组化法检测各组细胞总AKT蛋白及磷酸化AKT (p-AKT)表达水平。β半乳糖酶染色法染色后,倒置显微镜下观察,胞浆蓝染者为衰老细胞,随机取四个视野,计数阳性细胞数和细胞总数,取其平均值计算细胞衰老率。免疫组化法染色结果使用imagej图像处理软件测量灰度值。结果：1.不同浓度葡萄糖干预HUVEC 24h后,随着葡萄糖浓度增加,细胞衰老率增加。5.6mmol/L葡萄糖对内皮细胞衰老作用不明显。与5.6mmol/L葡萄糖组比较,16.7mmol/L和33mmol/L葡萄糖组均导致细胞衰老率的显著增加(P<0.01)。33mmol/L组与16.7mmol/L组相比,衰老率存在显著差异性(P<0.01)。各葡萄糖组总AKT蛋白表达水平变化无统计学意义(P>0.05)。p-AKT蛋白表达水平随着葡萄糖浓度的增加出现下调,并与细胞衰老呈负相关。16.7mmol/L和33mmol/L组与5.6mmol/L组比较,p-AKT表达明显下调(P<0.01)。33mmol/L组与16.7mmol/L组比较,p-AKT表达下调存在显著差异性(P<0.01)。2.不同浓度胰岛素干预HUVEC 24h后,各组细胞衰老率具有明显差异。与对照组比较,1nmol/L胰岛素组和10nmol/L胰岛素组细胞衰老率降低(P<0.01),且1nmol/L组细胞衰老率较10nmol/L组降低更明显(P<0.05)。与对照组和其他胰岛素组比较,100nmol/L胰岛素组细胞衰老率明显增加(P<0.01)。3.不同浓度胰岛素与对照组间,总AKT蛋白表达无差异(P>0.05)。与对照组相比,1nmol/L胰岛素组和10nmol/L胰岛素组p-AKT表达上调(P<0.01),1nmol/L组较10nmol/L组p-AKT表达上调更显著(P<0.01)。100nmol/L胰岛素组p-AKT表达与对照组比较,无统计学差异(P>0.05)。结论：1.高浓度葡萄糖可诱导人脐静脉内皮细胞衰老,随着浓度增加衰老程度加重,其机制可能与AKT蛋白活性下调有关。2.低浓度胰岛素可延缓内皮细胞衰老,可能与AKT蛋白活性上调有关,且其保护作用可能与降低葡萄糖浓度效应无关。3.高浓度胰岛素可导致血管内皮细胞衰老,其机制可能与AKT蛋白活化无关。更多还原

【Abstract】 Background and Objective:Cell senescence is one of the main causes of organism aging and diseases including atherosclerosis and hypertension. The prevalence of heart event and more severe heart problems increased obviously in patients with Diabetes mellitus. Nevertheless, the mechanism of how diabetes induces heart disease is unclear. This study intends to investigate the effects of glucose and insulin on the human umbilical vein endothelial cells (HUVECs) senescence in vitro, and to detect the activity of protein kinase B (PKB/AKT) in these cells under different doses of glucose and insulin condition. Hopefully, the results might be helpful to understand the cardiovascular effects of glucose and insulin, and to explore the mechanism of diabetes-induced atherosclerosis.Methods:Human umbilical vein endothelial cells were cultured in vitro with low glucose DMEM medium plus 10% fetal bovine serum.3-5 generation of these cells in logarithmic growth phase were used in study. The cultured human umbilical vein endothelial cells were divided into two parts which were treated with different doses of glucose and insulin respectively, (1) glucose group:5.6mmol/L,16.7mmol/L,33mmol/L; (2) insulin group: control, 1nmol/L, 10nmol/L, 100nmol/L. Each group of these cells was incubated with different concentrations of glucose and insulin respectively as mentioned previous for 24h. Then, each group of these cells was measured for cell senescent phenotype with SA-β-gal staining assay and expression of total AKT and phospho-AKT (p-AKT) protein with immunohistochemistry staining assay. After senescence-association SA-β-gal staining, cells were observed by inverted microscope, and positive and total cell number were counted in four different visual field, then the average of cellular senescence rate were calculated. Also, total AKT and p-AKT protein expression were analyzed by image manipulation to measure gray scale in each group of these cells.Results:1. Cell senescence rates were increased with the glucose concentrations. The data shows that as compared with group of 5.6mmol/L glucose which have no evident effect on human umbilical vein endothelial cells senescence, the rate of cells senescence increased obviously in the group of 16.7mmol/L and 33mmol/L glucose (P<0.01). Moreover, as compared with 16.7mmol/L glucose, the cellular senescence rate of 33mmol/L glucose group increased significantly (P<0.01). The level of total AKT expression in each glucose group gave similar results (P>0.05), while p-AKT expression was down-regulated with the increase of glucose concentrations. Compared to 5.6mmol/L glucose, the level of p-AKT expression in the presence of 16.7mmol/L and 33mmol/L of glucose decreased significantly (P<0.01), and the changes of p-AKT expression between 16.7mmol/L glucose and 33mmol/L glucose were obvious(P<0.01), which means that p-AKT expression might be negative correlation to cell senescence.2. Different cell senescence rate were observed in human umbilical vein endothelial cells in the presence of lnmol/L, 10nmol/L, 100nmol/L of insulin for 24h. The result shows that when compared to control group, the cellular senescence rate of lnmol/L and lOnmol/L insulin decreased (P<0.01), and the cellular senescence rate of lnmol/L insulin decreased obviously (P<0.01) as compared with 10nmol/L insulin. But the cellular senescence rate of 100nmol/L insulin group increased obviously(P<0.01) as compared with control, 1nmol/L and 10nmol/L insulin group.3. There is no difference of total AKT expression between control group and each insulin group (P>0.05). Compared to control group, p-AKT expression was up-regulated(P<0.01) in the presence of 1nmol/L and 10nmol/L of insulin. While p-AKT expression unchanged (P>0.05) in 100nmol/L insulin group as compared with control instead, even though the cellular senescence rate of this group increased obviously. Conclusion:1. High glucose might induce vascular endothelial cell senescence and which might be related to down-regulation of AKT activity.2. Low concentration insulin might inhibit human umbilical vein endothelial cells senescence and up-regulate the activity of p-AKT. And which might not be correlated to the glucose reduction effect of insulin.3. High dose of insulin might enhance cellular senescence, and which might be unrelated to the activation of p-AKT protein.更多还原