动态检测血管紧张素Ⅱ刺激后STAT3信号核转位及其与衰老的关系

Dynamic Observation of the Nucleus Translocation of STAT3 in Senescent Cells Stimulated with Angiotension Ⅱ

为了动态观察复制性衰老细胞中血管紧张素Ⅱ(Ang Ⅱ)激活人信号转导与转录活化因子3(hSTAT3)信号转导途径的核转位情况及该途径在细胞衰老过程中的变化。将载体pMS1-hSTAT3上的目的基因STAT3序列亚克隆到pEGFP-C3报告载体中,构建出pEGFP-hSTAT3质粒;选择人胚肺二倍体成纤维细胞WI-38细胞株进行细胞培养,通过脂质体(Effectene)转染的方法,将pEGFP-STAT3质粒分别转染至19代,42代WI-38细胞中;在激光共聚焦显微镜下动态观察血管紧张素Ⅱ激活STAT3的核转位的变化及不同代数的差别.结果显示AngⅡ刺激细胞后,出现活化的STAT3在胞核内聚集现象,并且在19代细胞中15分钟开始出现,30—45分钟左右时达到高峰;42代细胞中30分钟左右开始出现,50—60分钟左右达到高峰。因此我们认为Ang Ⅱ刺激WI-38细胞,能出现hSTAT3信号转导的核内集聚现象;并且随着WI-38细胞传代数的增加,STAT3信号转导入核时间延迟。说明随着细胞的复制性衰老,通过STAT3通路转导的信号活性逐渐降低,最终影响细胞的增殖,可能是促进细胞衰老的原因之一。

To investigate the dynamic nuclear-translocation change with aging of human signal transducer and activator of transcription 3 (STAT3) in normal human fetal lung diploid flbroblast cell line, WI-38, stimulated with angiotensin Ⅱ (10-6 mol/L), we constructed the plasmid pEGFP-hSTAT3 by subcloning of the hSTAT3 cDNA full sequence from pMS1-hSTAT3 into pEGFP-C3, following which transfections through the effectene were conducted into WI-38 cells of the 19th passage and 42nd passage, respectively, and then observed using the laser scanning confocal microscopy (LSCM). Our results revealed: (1) that after exposure to angiotensin Ⅱ, WI-38 cells took on an accumulation of STAT3 in the nucleus from the cytoplasms; (2) that in cells of the 19th passage, STAT3 nuclear accumulation began 15 and peaked 30-45 minutes after addition of angiotensin Ⅱ, while in the 42nd passage cells STAT3 nuclear accumulation began 30 and peaked 50-60 minutes after stimulation. Taken together, our results demonstrated that STAT3 nuclear transloca-tion were delayed by cellular senescence in WI-38 cells, which may interfere with the cell proliferation and be one of the mechanisms of aging.