摘要
目的观察细胞外信号调节激酶1/2(ERK1/2)在血管紧张素Ⅱ(AngⅡ)诱导的内皮细胞中不同时点的表达变化。方法制备AngⅡRPMI1640培养液(10-6 mol/L)培养人脐静脉内皮细胞,采用MTT测定内皮细胞存活率,β-gal染色、细胞周期分析鉴定细胞衰老,透射电子显微镜观察衰老细胞超微结构。细胞免疫化学染色法分析Bcl-2、Bax蛋白表达变化,Western印迹测定磷酸化ERK1/2水平。结果 AngⅡ诱导组存活的细胞数为对照组的〔(81.9±0.04)%,P<0.01)〕;约80%的细胞呈现β-gal阳性染色,流式细胞仪检测细胞周期停滞于G0～G1,证实细胞衰老;透射电子显微镜可见AngⅡ诱导组衰老的细胞体积较大,核形不规则,细胞核染色质浓缩和凝聚。与对照组相比,Bcl-2 mRNA表达呈持续性降低,Bcl-2/Bax比值下降,ERK1/2磷酸化水平于12 h明显增加,24 h达到高峰(P<0.01),36 h下降至稳定,总ERK1/2蛋白水平无明显变化。结论ERK1/2信号转导途径参与AngⅡ诱导内皮细胞衰老的发生、发展过程,并可能通过调控内皮细胞Bcl-2/Bax比值来实现。
Objective To explore the changes in extraeellular signal-regulated protein kinase （ERK1/2） in endothelial cell senes- cence induced by angiotensin Ⅱ at the different time courses, and its possible molecular mechanism. Methods Human umbilical vein endo- thelial cells （HUVECs） were cultured in vitro and intervened by Ang Ⅱ. HUVECs were divided into control and Ang Ⅱ groups （stimulated by Ang Ⅱ 10-6 mol/L for 48 h）. The cell living rate was observed by MYⅡ; lB-gal staining and cell cycle analysis were used to identify cell aging status. Cell senescence was used to studied by transmission electric microscopy. The expressions of apoptosis-assoeiation genes Bcl-2, Bax were detected by immunoeytochemistry and ERK1/2 levels were detected by Western-blotting at different time points. Results 10-6 mol/L Ang Ⅱ stimulated cell senescence. The cell living rate by Ang Ⅱ -induced cells was （ 81.9%± 4. 1% , P 〈 0. 01 ）, the positive cell number of β-gal staining was significantly higher in Ang Ⅱ -induced ceils than that in control cells ; the cell cycle was at Go - Gl , S phase and G2/M phase were a tendency to disappearance in Ang Ⅱ-induced cells, the senescent cells were significantly increased under transmis- sion electric microscopy. Bcl-2 mRNA level was time-dependently decreased, the radio of Bel-2/Bax was decreased markedly than that of control group （ P 〈 0. 05 ）. Phosphorylation of ERK1/2 was began to increase and reached the peak at 24 h（ P 〈 0.01 ）. Conclusions Cell senescence is possibly important factor for atherosclerosis. One of its molecular mechanisms might be associated with decreasing the expres- sion of Bcl-2 and the radio of Bcl-2/Bax. There is a probability that activated ERK1/2 signal pathway is involved in the process of pathologic and physiologic reaction in the senescence of endothelial cell induced by Angiotensin Ⅱ.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2012年第2期294-296,共3页
Chinese Journal of Gerontology
基金
国家973重点基础研究发展规划基金资助项目(No.2007CB507405)
中华医学会临床医学科研专项资金-动脉粥样硬化研究资金资助(No.09010530208)
辽宁省科学技术研究项目(No.20091104)
沈阳市科学技术计划项目(F10-205-1-44)
关键词
细胞外信号调节激酶
内皮细胞
动脉硬化
血管衰老
Extracellular signal-regulated protein kinase
Endothelial cell
Atherosclerosis
Vascular senescence
