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Rac1蛋白活化加速缺氧诱导的人血管内皮细胞衰老

韩雅玲, 于海波, 闫承慧, 康建, 孟子敏, 张效林, 李少华, 王士雯

沈阳军区总医院全军心血管病研究所心内科.辽宁,沈阳 110016；美国Robert Wood Johnson 医学院病理实验科.新泽西州 08854；解放军总医院老年心血管病研究所.北京 100853

摘要

为阐明Rac1蛋白在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)衰老中的作用及分子机制,作者采用持续缺氧的方法诱导内皮细胞衰老,检测缺氧前后内皮细胞衰老标志基因SA--#beta#--Gal和PAI--1的表达、细胞周期分布和细胞增殖情况,同时分析缺氧前后细胞内Rac1蛋白的表达。结果显示,持续缺氧96h后,HUVECs体积变大,细胞浆内颗粒和空泡增多,SA--#beta#--Gal活性明显增加,PAI--1基因表达升高,细胞发生G_(1)期阻滞,细胞增殖受抑,活化型Rac1蛋白表达上调,提示持续缺氧诱导的内皮细胞衰老可能与Rac1蛋白的活化有关。为进一步明确内皮细胞衰老与Rac1蛋白的关系,应用逆转录病毒将持续活化型Rac1(v12Rac1)和主导抑制型Rac1(N17Rac1)基因分别瞬时感染HUVECs,比较种HUVECs(HUVECs,V12Rac1--HUVECs,N17Rac1--HUVECs)缺氧后的衰老变化,并分析其下游调控分子-----血清反应因子(serum response factor,SRF)的表达和定位变化。研究发现,缺氧培养V12Rac1--HUVECs 48h即可引起细胞衰老，表现为SA--#beta#--Gal活性明显增加，PAI-1基因表达升高，细胞出现明显的G_(1)期阻滞并且细胞增殖受抑；其改变与缺氧96h的HUVECs相似；而N17Rac1明显抑制缺氧引起的内皮细胞衰老发生。上述结果说明Rac1蛋白活化可以加速缺氧诱导的内皮细胞衰老，而抑制Rac1蛋白的活性则可抑制缺氧诱导的内皮细胞衰老。为进一步研究Rac1蛋白引起内皮细胞衰老的机制，通过免疫荧光染色及Western blot分析检测三种细胞缺氧处理后SRF的表达发现：与HUVECs细胞比较，V12Rac1引起缺氧48h HUVECs 核蛋白中SRF的表达明显下降， SRF入核转位受到明显抑制；而N17Rac1感染后，缺氧HUVECs细胞核蛋白中SRF表达明显增多。上述结果提示：缺氧状态下Rac1蛋白活化能够明显加速HUVECs衰老，而抑制Rac1蛋白活性则明显抑制缺氧诱导的HUVECs衰老, SRF蛋白的核转位活化参与了Rac1蛋白调控HUVECs衰老的发生。

关键词： Racl; 缺氧; 内皮细胞; 衰老

Racl accelerates endothelial cell senescence induced by hypoxia {sl in vitro}

Han Yaling, Yu Haibo, Yan Chenghui, KANG Jian, Meng Zimin, Zhang Xiaolin, Li Shaohua, Wang Shiwen

Department of Cardiology, General Hospital of Shenyang,The Institute of Cardiovascular Research, PLA.Shenyang 110016,Liaoning；USA；Institute of Geriatric Cardiology, General Hospital of PLA.Beijing 100853

Abstract

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cells (HUVECs) senescence，we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-#beta#-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-#beta#-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G_(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96h. Accompanied with these changes, activated Rac1 expression increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVECs senescence, HUVECs were transiently infected with the constitutively active forms of Rac1 (V12Rac1) or dominant negative forms of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs,V12Rac1-HUVECs) after hypoxia for 48h and 96h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-#beta#-Gal activation, PAI-1 expression increase, G_(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96h hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96h. All the results identified that the activation of Rac1 might accelerate HUVECs senescence induced by hypoxia and that inactivation of Rac1 could block partly the cells senescence. To further investigate the mechanism of HUVECs senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggested that activation of Rac1 accelerated endothelial cells senescence and inhibition of Rac1 activity prevented HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.

Key words: Rac 1;Hypoxia;Endothelial cell;senescence

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韩雅玲, 于海波, 闫承慧, 康建, 孟子敏, 张效林, 李少华, 王士雯. Rac1蛋白活化加速缺氧诱导的人血管内皮细胞衰老[J]. 生理学报 2006; 58 (3): .

Han Yaling, Yu Haibo, Yan Chenghui, KANG Jian, Meng Zimin, Zhang Xiaolin, Li Shaohua, Wang Shiwen. Racl accelerates endothelial cell senescence induced by hypoxia {sl in vitro}. Acta Physiol Sin 2006; 58 (3): (in Chinese with English abstract).