葡萄糖和胰岛素对血管内皮细胞衰老的影响及机制研究
发布时间：2018-08-26 12:14
【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(P0.01)。33mmol/L组与16.7mmol/L组相比,衰老率存在显著差异性(P0.01)。各葡萄糖组总AKT蛋白表达水平变化无统计学意义(P0.05)。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(P0.01)。33mmol/L组与16.7mmol/L组比较,p-AKT表达下调存在显著差异性(P0.01)。 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(P0.01),且1nmol/L组细胞衰老率较10nmol/L组降低更明显(P0.05)。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(P0.01)。 3.不同浓度胰岛素与对照组间,总AKT蛋白表达无差异(P0.05)。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(P0.01),1nmol/L组较10nmol/L组p-AKT表达上调更显著(P0.01)。100nmol/L胰岛素组p-AKT表达与对照组比较,无统计学差异(P0.05)。 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。
[Abstract]:Background & AIM: cellular aging promotes aging and aging related diseases such as atherosclerosis, hypertension and so on. The incidence of cardiac events and the severity of disease in diabetic patients are increased, but the mechanism is not clear. The effects of glucose and insulin at different concentrations on (human umbilical vein endothelial cell, HUVEC) senescence and activation of protein kinase B (protein kinase BPKB / AKT in human umbilical vein endothelial cells (HUVECs) were studied in vitro. In order to understand the effect of glucose and insulin on cardiovascular, and further study the pathogenesis of diabetes-induced atherosclerosis. Methods: human umbilical vein endothelial cells were cultured in vitro on DMEM medium containing 10% fetal bovine serum. The experiment was divided into three groups: (1) glucose intervention group: different concentrations of glucose, including 5.6 mmol / L ~ (16. 7) mmol / L ~ (33) mmol / L group; (2) insulin group: control group: 1 nmol / L ~ (10) nmol / L ~ (-1) nmol / L group. The cells in each group were cultured to logarithmic growth stage and treated with corresponding concentration for 24 h. The senescence rate of the cells was detected by 尾 -galactozyme staining. The expression of total AKT protein and phosphorylated AKT (p-AKT) were detected by immunohistochemical method. After 尾 -galactozyme staining, the cytosolic blue stained cells were aged cells, and four visual fields were randomly selected. The number of positive cells and the total number of cells were counted, and the average cell senescence rate was calculated. The results of immunohistochemical staining were measured by imagej image processing software. Results: 1. With the increase of glucose concentration, the senescence rate of endothelial cells increased by 5.6mmol / L glucose for 24 hours after different concentrations of glucose were treated with HUVEC, and the senescence effect of glucose on endothelial cells was not obvious. Compared with 5.6mmol/L glucose group, 16.7 mmol / L group and 33mmol/L glucose group significantly increased the senescence rate of cells (P0.01) .33mmol / L group compared with 16.7mmol/L group, there was significant difference in senescence rate (P0.01). There was no significant change in the expression of total AKT protein in each glucose group (P0.05). The expression of p-AKT protein decreased with the increase of glucose concentration. The expression of p-AKT in 33mmol/L group was significantly lower than that in 5.6mmol/L group (P0.01) .33mmol / L group and 16.7mmol/L group (P0.01). After 24 hours of insulin treatment with different concentrations of insulin, the senescence rate of the cells in each group was significantly different. Compared with the control group, the cell senescence rate of 1nmol / L insulin group and 10nmol/L insulin group was decreased (P0.01), and the senescence rate of 1nmol/L group was significantly lower than that of 10nmol/L group (P0.05). Compared with the control group and other insulin groups, the senescence rate of 100 nmol / L insulin group was significantly increased (P0.01). There was no difference in total AKT protein expression between different concentrations of insulin and control group (P 0.05). Compared with the control group, there was no significant difference in the expression of p-AKT between the 1 nmol / L insulin group and the 10nmol/L insulin group (P0.01) and 1 nmol / L group compared with the control group (P0.01). 100 nmol / L insulin group (P0.01) and the control group (P0.05). Conclusion: 1. High concentration of glucose could induce the senescence of human umbilical vein endothelial cells, and the mechanism might be related to the down-regulation of AKT protein activity. Low concentration of insulin can delay the aging of endothelial cells, which may be related to the up-regulation of AKT protein activity, and its protective effect may not be related to the effect of decreasing glucose concentration. 3. High concentration of insulin can lead to aging of vascular endothelial cells, its mechanism may not be related to the activation of AKT protein.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2204837
[Abstract]:Background & AIM: cellular aging promotes aging and aging related diseases such as atherosclerosis, hypertension and so on. The incidence of cardiac events and the severity of disease in diabetic patients are increased, but the mechanism is not clear. The effects of glucose and insulin at different concentrations on (human umbilical vein endothelial cell, HUVEC) senescence and activation of protein kinase B (protein kinase BPKB / AKT in human umbilical vein endothelial cells (HUVECs) were studied in vitro. In order to understand the effect of glucose and insulin on cardiovascular, and further study the pathogenesis of diabetes-induced atherosclerosis. Methods: human umbilical vein endothelial cells were cultured in vitro on DMEM medium containing 10% fetal bovine serum. The experiment was divided into three groups: (1) glucose intervention group: different concentrations of glucose, including 5.6 mmol / L ~ (16. 7) mmol / L ~ (33) mmol / L group; (2) insulin group: control group: 1 nmol / L ~ (10) nmol / L ~ (-1) nmol / L group. The cells in each group were cultured to logarithmic growth stage and treated with corresponding concentration for 24 h. The senescence rate of the cells was detected by 尾 -galactozyme staining. The expression of total AKT protein and phosphorylated AKT (p-AKT) were detected by immunohistochemical method. After 尾 -galactozyme staining, the cytosolic blue stained cells were aged cells, and four visual fields were randomly selected. The number of positive cells and the total number of cells were counted, and the average cell senescence rate was calculated. The results of immunohistochemical staining were measured by imagej image processing software. Results: 1. With the increase of glucose concentration, the senescence rate of endothelial cells increased by 5.6mmol / L glucose for 24 hours after different concentrations of glucose were treated with HUVEC, and the senescence effect of glucose on endothelial cells was not obvious. Compared with 5.6mmol/L glucose group, 16.7 mmol / L group and 33mmol/L glucose group significantly increased the senescence rate of cells (P0.01) .33mmol / L group compared with 16.7mmol/L group, there was significant difference in senescence rate (P0.01). There was no significant change in the expression of total AKT protein in each glucose group (P0.05). The expression of p-AKT protein decreased with the increase of glucose concentration. The expression of p-AKT in 33mmol/L group was significantly lower than that in 5.6mmol/L group (P0.01) .33mmol / L group and 16.7mmol/L group (P0.01). After 24 hours of insulin treatment with different concentrations of insulin, the senescence rate of the cells in each group was significantly different. Compared with the control group, the cell senescence rate of 1nmol / L insulin group and 10nmol/L insulin group was decreased (P0.01), and the senescence rate of 1nmol/L group was significantly lower than that of 10nmol/L group (P0.05). Compared with the control group and other insulin groups, the senescence rate of 100 nmol / L insulin group was significantly increased (P0.01). There was no difference in total AKT protein expression between different concentrations of insulin and control group (P 0.05). Compared with the control group, there was no significant difference in the expression of p-AKT between the 1 nmol / L insulin group and the 10nmol/L insulin group (P0.01) and 1 nmol / L group compared with the control group (P0.01). 100 nmol / L insulin group (P0.01) and the control group (P0.05). Conclusion: 1. High concentration of glucose could induce the senescence of human umbilical vein endothelial cells, and the mechanism might be related to the down-regulation of AKT protein activity. Low concentration of insulin can delay the aging of endothelial cells, which may be related to the up-regulation of AKT protein activity, and its protective effect may not be related to the effect of decreasing glucose concentration. 3. High concentration of insulin can lead to aging of vascular endothelial cells, its mechanism may not be related to the activation of AKT protein.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
【参考文献】
相关期刊论文 前7条
1 李围;杨晓明;;激酶调控PKB/Akt活性的机制[J];医学分子生物学杂志;2007年04期
2 刘海涛;张海锋;司瑞;张全江;张昆茹;郭文仪;王海昌;高峰;;胰岛素保护缺血/再灌注心脏:PI3-K/Akt和JNKs信号通路间的交互作用(英文)[J];生理学报;2007年05期
3 周颖;王建;贺福初;;蛋白激酶B(PKB/Akt)的结构、调控与功能[J];生命的化学;2006年03期
4 马向红;黄体钢;杨万松;周丽娟;刘洪梅;;高糖和胰岛素对内皮细胞产生一氧化氮的影响[J];天津医药;2006年08期
5 陆庆明;邸春霞;王海昌;张荣庆;;自发性高血压大鼠骨髓来源内皮祖细胞数量、功能及吡格列酮的预处理[J];中国组织工程研究与临床康复;2008年29期
6 单海燕;白小涓;张四扬;陈香美;;AngⅡ诱导血管内皮细胞衰老及凋亡相关基因的表达[J];中国病理生理杂志;2008年06期
7 彭艳华;王跃;;细胞衰老与机体老化关系的研究进展[J];中国老年学杂志;2008年09期
，本文编号：2204837
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2204837.html
最近更新
教材专著
