Object and BackgroundVascular endothelial cell (VEC) is one of the most important cell colonies in the body, which has lots of functions, such as secretion, synthesis, metabolism, immunity and so on. It widely distributes in the body and forms the interface between blood and tissue, not only relates to angiotasis, vasopermeability and blood coagulation, but also has a critical role in the inflammation by producting proinflammatory cytokines interact with the leukocytes in the circulation. The lesion of VEC is an important tissue damage response along with the inflammation. Most of inflammatory factors producted by macrophages, lymphocytes, neutrophils and VECs themselves, such as tumor necrosis factor-a (TNF-a), interferon γ (DFNγ), interleukin-1β (IL-1β) can cause the lesion of VEC, which inevitably result in endothelial functional disorder that the production of nitric oxide (NO) is abnormal. NO is a free radical synthesized by nitricoxide synthase (NOS) from L-Arginine, which serves as a key signalling molecule in physiological processes such as vasodilation, anti-platelet aggregation, inhibits the proliferation of the smooth muscle cells and so on. However, so much NO producted in the inflammatory diseases for the induction of cytokines may promote apoptosis and result in the lesion of VEC .NOS is the rate-limiting enzyme of NO, whose enzymatic activity and expression directly affect the production of NO. There are three subtypes of NOS definited in biological tissue: neuron NOS (nNOS or NOS1), endothelial NOS (eNOS or NOS3) and inducible NOS (iNOS or NOS2). The former two are also called constitution NOS (cNOS). The definitive mechanism how TNF-α affects the apoptosis or necrosis of the endothelial cells is not clear. But it can inhibit eNOS, activate iNOS and then affect the synthesis and release of NO, which may play an important role in its cytotoxic effect to tissue damage. The effect of TNF-α on the production of NO in endothelial cells is still a controversy, because the source of EC or the dosage of TNF-α in different study was not same, the results of NO with TNF-α were not same either. In those studies, human umbilical vein endothelial cells (HUVEC) were rarely used to investigate the association of TNF-α and NO and NOS. In our study, we detected the activity of eNOS and the production of NO in HUVEC and supernatant after treatment with different concentration of TNF-α.for different times, thus discussed the influence of TNF-α on NO and the molecular mechanism of regulation of NOS.Materials and MethodsHuman EC were obtained from umbilical vein (HUVEC) and cultured aspreviously described. Routinely HUVECs were digested by 0.1% type I collagenase from umbilical vein at 37°C for about 30min.Then these cells were plated in 24-well plate at a density of 2X 105/mL in Ml 99 medium containing 15% fetal bovine serum (Hyclone, Australia) supplemented with endothelial growth factor (10 ug/mL, R