切应力诱导内皮细胞eNOS基因表达的信号调控机制研究

It was showed that endothelial cells not only play an important role in the physiological process including blood pressure regulation, permeability of blood vessels, prevention of leukocyte adhesion and aggregation of blood platelet, but also involve in pathogenesis and development of shock, hypertension, atherosclerosis, ischemia reperfusion injury, etc. Nitric oxide (NO) is an important regulator of endothelial cell function. In endothelial cells, No is produced by the catalysis of endothelial nitric oxide synthase (eNOS) on L-arginine. Being different from inducible nitric oxide synthase (iNOS) on the regulation of its expression and function, eNOS is a constitutively expressed NOS (cNOS). For a long time, it was taken for granted that the regulation of NO production is mainly regulated by the control of iNOS expression, while the NO production catalyzed by cNOS is regulated by the change of enzyme activity. However, it was found recently that eNOS is not constitutively expressed with a stable level, but with the expression regulated by many physiological factors. It was proved that shear stress on the blood vessel wells could induce the binding activity of shear stress response element (SSRE) and eNOS gene expression by increase the promoter transactivity of eNOS which would bring about the NO production in endothelial cells. eNOS is very important for many physiological functions of human body, furthermore, its abnormal production participates a lot of pathological processes. .For the study of gene expression mechanism of eNOS in endothelial cells, a red fluorescent protein (RFP) reporter gene vector (pDseNOSRed) containing human eNOS promoter that may express in mammalian cells was constructed. For the functional study of different region of eNOS promoter, the plasmid vectors (pDsF1033Red，pDsF494Red and pDsF166Red) containing different regions of human eNOS promoter coupled to a RFP reporter gene that may express in mammalian cells were constructed. All these constructs were proved to be correct by double restriction enzyme digestion, PCR and sequencing. Following the transfection of HEK293 or NIH3T3 cells with these report gene constructs, the cells were observed for the expression and distribution of fluorescence under fluorescent microscope. It was found that the red fluorescent emitted by a red fluorescent protein dispersed all over the cells, appearing at 12 h after transfection, reaching peak at 36-48 h and almost disappearing at 120 h. In NIH3T3 cells, 95 % of the red fluorescent emitted by a red fluorescent protein dispersed all over the cells, appearing at 48~60 h after transfection, reaching peak at 96~144 h, becoming strongest in light at 144 h, gradually disappearing after 168 h and remaining little red fluorescence in 21 days. The quantity and intensity in expressions of RFP drived by different regions of human eNOS promoter were clearly lower than by a strong promoter, PCMV IE. The red fluorescent protein reporter gene vectors containing human eNOS promoter or different regions of human eNOS promoter were successfully constructed. These recombinant vectors may efficaciously express in mammalian cells, appearing not strong transcriptional activities, which provide practical and feasible tools for the functional study of gene expression regulation of human eNOS. It was found from the study of regulation of eNOS promoter transactivity that the basic transactivity of eNOS promoter is mainly regulated by SP1, but not SSRE. The proinflammatory factors, both LPS and TNF-α, could down-regulate the transactivity of eNOS promoter in endothelial cells, while transform growth factor-β (TGF-β) could up-regulate the transactivity of eNOS promoter. All these results suggest that eNOS in endothelial cells is not only a substance that was traditionally taken as a specific responsive factor for the physiological shear stress on the blood vessel wells, but also has a good response to some inflammatory mediators. This important finding suggests that the regulation of gene expression of eNOS in endothelial cells is invo

应用生物力学、分子和细胞生物学的方法研究切应力对内皮细胞ｅＮＯＳ基因表达、ＮＯ生成以及ＭＡＰＫ激酶活性的影响；不同ＭＡＰＫ通路对ｅＮＯＳ启动子转录活性的调控及其与切应力么应元件之间的关系。确立内皮细胞对切应力发生反应引起ｅＮＯＳ基因表达的信号转导路线。血流切应力诱导ｅＮＯＳ基因表达参与多种血管功能障碍的病理和生理过程，其研究有着重要科学意义。