摘要
目的:以TNF-α/act D诱导的细胞凋亡作为一种内皮细胞损伤的模型,通过转染细胞色素P450表氧化酶基因BM3.F87V、2C11OR以及2J2产生内源性EDHF(EETs),探讨EDHF是否具有抗内皮细胞损伤的作用及其机制。方法:以CYP450表氧化酶BM3.F87V、2C11OR及2J2基因转染3-4代培养的牛主动脉血管内皮细胞。通过MTT方法检测细胞活性。基因转染24 h后,用TNF-α/act D作用一定时间诱导内皮细胞凋亡,以细胞形态学观察,DNA ladder分析,流式细胞仪分析(Annexin-V-FITC和propidium iodide双染法)作为检测细胞凋亡的指标。结果:MTT分析显示,转染CYP450 BM3.F87V、2C11OR以及CYP2J2基因的内皮细胞活性(分别为189.0%±18.0%,166.0%±22.6%或131.0%±22.8%)强于对照组(100.0%±21.3%)。经TNF-α/act D诱导,转染pCB6的对照组内皮细胞大量脱落,未脱落细胞中许多细胞核呈碎裂颗粒状(约20.1%±0.9%);而转染CYPBM3.F87V、CYP2C11OR或CYP2J2基因的内皮细胞仅见少数核呈碎颗粒状(分别约1.2%±0.2%,1.0%±0.2%或3.1%±0.2%)。DNA ladder分析显示TNF-α/act D诱导后,转染表氧化酶基因的内皮细胞所形成的梯形条带弱于对照组。进一步用流式细胞仪定量检测表明,转染细胞色素表氧化酶基因CYP BM3F87V或2C11OR内皮细胞中凋亡细胞所占比例(分别为12.35%±4.55%,10.92%±3.57%)明显低于对照组(29.57%±2.84%),而转染CYP2J2的细胞凋亡率也明显低于对照组。结论:转染CYP450基因能够增加内皮细胞活性,抑制TNF-α诱导的内皮细胞凋亡,具有内皮细胞保护效应。
AIM: To investigate whether endogenous endothelium -derived hyperpelarizing factors (EHDFs) produced by CYP epoxygenases BM3 · F87V, 2CllOR or CYP2J2 transfection was able to protect endothelial cells against apoptosis induced by tumor necrosis factor alpha. METHODS : Three or four passages of cultured bovine aortic endothelial cells (BAECs) were transfected with epoxygenases or the empty vector ( pCB6 ). Cell viability was detected by MTT assay. Apoptosis of transfected endothelial cells was evaluated by DNA ladder assay, flow cytometry and morphological observations under fluorescence microscopy. RESULTS: Overexpression of CYP epoxygenases BM3· F87V, 2CllOR, CYP2J2 increased cell viability respectively observed by MTT assay. The percentage of cells undergoing apeptosis was decreased in 2C11OR-, BM3F87V- or 2J2 -transfected cells compared to the vector as evaluated by DNA fragment assay, flow cytometry analysis and morphological observations under fluorescence microscopy. CONCLUSION: Overexpression of CYP epexygenases BM3 · F87V, 2 C11 OR or 2.J2 increases cell viability and protects endothelial cells against TNF -α - induced apeptosis. These findings suggest new targets to investigate the endothelium - associated disorders and provide novel thera-peutic strategies to treat them by modulating cytochrome P450 epoxygenases.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第10期1878-1882,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.39870307)
关键词
内皮源性超极化因子
内皮细胞
环氧二十碳烯酸
细胞凋亡
Endothelium - derived hyperpolarizing factor
Endothelial cells
Epoxyeicosatrienoic acids
Apoptosis