细胞色素P450花生四烯酸表氧化酶抑制TNF-α诱导的内皮细胞凋亡

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。

Arachidonic acid cytochrome P450 epoxygenases protect vessel endothelial cells against apoptosis induced by tumor necrosis factor-α

AIM: To investigate whether endogenous endothelium-derived hyperpolarizing factors (EHDFs) produced by CYP epoxygenases BM₃·F87V,2C11OR 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,2C11OR,CYP2J2 increased cell viability respectively observed by MTT assay.The percentage of cells undergoing apoptosis 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 epoxygenases BM₃·F87V,2C11OR or 2J2 increases cell viability and protects endothelial cells against TNF-α-induced apoptosis.These findings suggest new targets to investigate the endothelium-associated disorders and provide novel therapeutic strategies to treat them by modulating cytochrome P450 epoxygenases.