论文部分内容阅读
血管内皮细胞富含细胞色素P450表氧化酶,它代谢花生四烯酸产生内皮源性超极化因子,(EDHF,即EETs),它与血管内皮细胞产生的一氧化氮(NO)之间的关系尚不清楚,经转基因技术在原代培养的牛主动脉内皮细胞中产生内源性EDHF,并研究了EDHF对NO合成的关键酶——内皮NO合酶(eNOS)基因表达的调节作用,将表氧化酶基因CYP BM_3F87V克隆于真核表达载体pCB_6,转染了经3～4代培养的牛主动脉内皮细胞,产生高浓度的内源性14,15-EET.采用Western blot和Northern blot分别从蛋白质水平和RNA水平判断内源性14,15-EET对eNOS基因表达和转录的影响,同时根据[~3H]L-精氨酸转变为[~3H]L-瓜氨酸的水平来判断其对eNOS活性的影响。结果显示:与转染pCB_6的对照细胞相比,转染的pCB_6-BM_3F87V在内皮细胞中过度表达后能显著上调eNOS的蛋白质表达和eNOS的mRNA水平,明显增加了eNOS的活性,因此,转染表氧化酶BM_3F87V对eNOS基因有显著上调作用。这提示细胞色素P450表氧化酶能够通过促进内皮细胞内EDHF的产生来调节NO生成,进而改善内皮细胞功能,这可能为治疗心血管疾病提供了一种新思路。
Vascular endothelial cells are rich in cytochrome P450 epoxidase, which metabolizes arachidonic acid to produce endothelium-derived hyperpolarizing factors (EDHF, or EETs) that interact with nitric oxide (NO) produced by vascular endothelial cells It is unclear that the endogenous EDHF is produced in primary cultured bovine aortic endothelial cells by transgenic technology and the regulatory effect of EDHF on the expression of endothelial NO synthase (eNOS), a key enzyme in NO synthesis, is studied Epoxygenase gene CYP BM_3F87V was cloned into eukaryotic expression vector pCB_6 and transfected into bovine aortic endothelial cells cultured for 3 to 4 generations to generate high concentration of endogenous 14,15-EET. Western blot and Northern blot Judging the effect of endogenous 14,15-EET on eNOS gene expression and transcription from protein level and RNA level, and judging from the level of [~ 3H] L-arginine into [~ 3H] L-citrulline Its effect on eNOS activity. The results showed that transfection of pCB_6-BM_3F87V significantly up-regulated eNOS protein expression and eNOS mRNA level in endothelial cells after transfection compared with control cells transfected with pCB_6 significantly increased eNOS activity. Therefore, transfection Epoxide oxidase BM_3F87V significantly up-regulated the eNOS gene. This suggests that cytochrome P450 epoxide oxidase regulates NO production by promoting the production of EDHF in endothelial cells, thereby improving endothelial cell function, which may provide a new idea for the treatment of cardiovascular diseases.