摘要
背景:目前研究证实,Rac1蛋白能够调控甘油醛-3-磷酸脱氢酶氧化酶的表达,刺激内源性活性氧产生,引起内皮细胞毒性改变。目的:探讨Rac1蛋白在缺氧诱导的人血管内皮细胞凋亡中的作用及其调控机制。设计、时间及地点:单一样本观察,于2007-01/2008-05在解放军沈阳军区总医院医学实验科完成。材料:自备人脐静脉内皮细胞、Phoenix amphotropic 293包装细胞、持续活化型pLNCX-L61Rac1和主导抑制的pLNCX-N17Rac1反转录病毒真核表达载体。方法:体外培养人脐静脉内皮细胞,将细胞以体积分数为1%O2、5%CO2及94%N2缺氧条件下培养72h。用持续活化型pLNCX-L61Rac1(L61Rac1感染组)和主导抑制型pLNCX-N17Rac1(N17Rac1感染组)反转录病毒真核表达载体转染人脐静脉内皮细胞,并筛选稳定表达的细胞克隆。主要观察指标:①感染后稳定表达阳性克隆筛选。②采用pull-down和Western blot分析感染前后细胞内Racl的活化。③采用持续缺氧的方法诱导内皮细胞凋亡,用TUNEL染色和Western blot检测缺氧72h后各实验组细胞中cleave-caspase3表达。④应用免疫荧光和Western blot分析血清反应因子的表达和定位。结果:筛选出稳定表达持续活化型pLNCX-L61Rac1和主导抑制型的pLNCX-N17Rac1的人脐静脉内皮细胞克隆;应用pull down和Western blot证实各组细胞内Racl的活化改变;与人脐静脉内皮细胞和N17Rac1感染细胞比较,缺氧72h后L61Rac1细胞发生明显凋亡。进一步应用Western blot分析证实3组细胞中血清反应因子表达无明显改变,但人脐静脉内皮细胞和N17Rac1-HUVECs中血清反应因子为入核表达,而L61Rac1-HUVECs中血清反应因子蛋白在细胞核周大量表达,血清反应因子发生出核转位。结论:Rac1参与了内皮细胞凋亡的调控,机制可能与其调控血清反应因子蛋白核转位有关。
BACKGROUND： It is confirmed that Racl regulates the expression of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, produces the endogenous reactive oxygen and results in the apoptosis of cells. OBJECTIVE： To investigate the effect of Racl protein in apoptosis of human umbilical vein endothelial cells （HUVECs） and its regulatory mechanism. DESIGN, TIME AND SETTING： A single sample observation was performed at the Department of Medical Experiment, General Hospital of Shenyang Military Area Command of Chinese PLA from January 2007 to May 2008. MATERIALS： HUVEC, Phoenix amphotropic 293 package cell, retrovirus vector pLNCX-L61Rac1 and pLNCX-N17Rac1. METHODS： HUVECs were cultured in vitro in hypoxic condition （1% O2, 5% CO2, or 94% N） for 72 hours. Then cells were infected with the constitutively active form of Racl （L61 Racl ） or dominant negative form of Racl （NI7Racl） using retrovirus vector pLNCX- L61Rac1 or pLNCX-NI7Racl, cell clones with stable expression was selected. MAIN OUTCOME MEASURES： ①Selection of positive cell clones. ②Activation of Racl was analyzed prior to and after infection with pull-down and Western blot. ③Expression of cleave-caspase3 was detected by TUNEL staining and Western blot at hours after hypoxia.④Expression and localization of SRF was determined by immunofluorescence and Western blot. RESULTS： Cell clones of pLNCX- L61Rac1 and pLNCX-N17Rac1 were screened successfully. Activation changes of Racl in each group were identified by pull down and Western blot. Compared to HUVECs and N17Rac1-HUVECs, there were more apoptotic cells were found in L61 Racl after hypoxia for 72 hours. Western blot demonstrated that the expression of SRF had no obvious changes in each group. However, it was expressed in nuclei of HUVECs and N17Rac1-HUVECs, which expressed around the nuclei in L61Racl- HUVECs with nuclear translocation. CONCLUSION： Racl is involved in the regulation of HUVECs apoptosis, which may be associated with nuclear translocation o
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第20期3853-3856,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
辽宁省科技攻关计划资助项目(2005225013-15)~~
