葡萄糖和胰島素對(duì)血管內(nèi)皮細(xì)胞衰老的影響及機(jī)制研究
發(fā)布時(shí)間：2018-08-26 12:14
【摘要】： 背景和目的：細(xì)胞衰老促進(jìn)機(jī)體老化和老年相關(guān)疾病如動(dòng)脈粥樣硬化、高血壓等發(fā)生。糖尿病患者的心臟事件發(fā)生率增加,病變程度加重,但其發(fā)生機(jī)制還不明確。本文探討體外培養(yǎng)條件下,不同濃度的葡萄糖和胰島素對(duì)人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cell, HUVEC)衰老及蛋白激酶B(protein kinase B,PKB/AKT)活化的影響。以期了解葡萄糖和胰島素對(duì)心血管的作用,并進(jìn)一步研討糖尿病致動(dòng)脈粥樣硬化的發(fā)病機(jī)制。 方法：用含10%胎牛血清的DMEM低糖培養(yǎng)基行體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞,取其對(duì)數(shù)生長期細(xì)胞用于實(shí)驗(yàn)。實(shí)驗(yàn)分組：(1)葡萄糖干預(yù)組：不同濃度的葡萄糖,包括5.6mmol/L、16.7mmol/L、33mmol/L組；(2)胰島素組：對(duì)照組、1nmol/L、10nmol/L、100nmol/L組。各組細(xì)胞培養(yǎng)至對(duì)數(shù)生長期,分別給與相應(yīng)濃度的處理因素,作用24h,用β半乳糖酶染色法檢測(cè)細(xì)胞衰老率,并用免疫組化法檢測(cè)各組細(xì)胞總AKT蛋白及磷酸化AKT (p-AKT)表達(dá)水平。β半乳糖酶染色法染色后,倒置顯微鏡下觀察,胞漿藍(lán)染者為衰老細(xì)胞,隨機(jī)取四個(gè)視野,計(jì)數(shù)陽性細(xì)胞數(shù)和細(xì)胞總數(shù),取其平均值計(jì)算細(xì)胞衰老率。免疫組化法染色結(jié)果使用imagej圖像處理軟件測(cè)量灰度值。 結(jié)果： 1.不同濃度葡萄糖干預(yù)HUVEC 24h后,隨著葡萄糖濃度增加,細(xì)胞衰老率增加。5.6mmol/L葡萄糖對(duì)內(nèi)皮細(xì)胞衰老作用不明顯。與5.6mmol/L葡萄糖組比較,16.7mmol/L和33mmol/L葡萄糖組均導(dǎo)致細(xì)胞衰老率的顯著增加(P0.01)。33mmol/L組與16.7mmol/L組相比,衰老率存在顯著差異性(P0.01)。各葡萄糖組總AKT蛋白表達(dá)水平變化無統(tǒng)計(jì)學(xué)意義(P0.05)。p-AKT蛋白表達(dá)水平隨著葡萄糖濃度的增加出現(xiàn)下調(diào),并與細(xì)胞衰老呈負(fù)相關(guān)。16.7mmol/L和33mmol/L組與5.6mmol/L組比較,p-AKT表達(dá)明顯下調(diào)(P0.01)。33mmol/L組與16.7mmol/L組比較,p-AKT表達(dá)下調(diào)存在顯著差異性(P0.01)。 2.不同濃度胰島素干預(yù)HUVEC 24h后,各組細(xì)胞衰老率具有明顯差異。與對(duì)照組比較,1nmol/L胰島素組和10nmol/L胰島素組細(xì)胞衰老率降低(P0.01),且1nmol/L組細(xì)胞衰老率較10nmol/L組降低更明顯(P0.05)。與對(duì)照組和其他胰島素組比較,100nmol/L胰島素組細(xì)胞衰老率明顯增加(P0.01)。 3.不同濃度胰島素與對(duì)照組間,總AKT蛋白表達(dá)無差異(P0.05)。與對(duì)照組相比,1nmol/L胰島素組和10nmol/L胰島素組p-AKT表達(dá)上調(diào)(P0.01),1nmol/L組較10nmol/L組p-AKT表達(dá)上調(diào)更顯著(P0.01)。100nmol/L胰島素組p-AKT表達(dá)與對(duì)照組比較,無統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論： 1.高濃度葡萄糖可誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞衰老,隨著濃度增加衰老程度加重,其機(jī)制可能與AKT蛋白活性下調(diào)有關(guān)。 2.低濃度胰島素可延緩內(nèi)皮細(xì)胞衰老,可能與AKT蛋白活性上調(diào)有關(guān),且其保護(hù)作用可能與降低葡萄糖濃度效應(yīng)無關(guān)。 3.高濃度胰島素可導(dǎo)致血管內(nèi)皮細(xì)胞衰老,其機(jī)制可能與AKT蛋白活化無關(guān)。
[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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363
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