| BackgroundAtherosclerosis (AS) is one of the major complications of chronic renal failure (CRF). The incidence of AS in CRF is markedly higher than that in general population and the age of incidence is shortened to 30-40 years. So AS in CRF has benn termed "accelerated AS" (AAS). The mechanism of the development of AAS in CRF is not clear yet. Previous study showed oxidative stress, resulting from a disruption of the natural balance between pro-and anti-oxidant systems has been occurred in CRF patients. In hemodialysis patients, recurrent blood interaction with bioincompatible dialysis membranes or contaminated endotoxin in dialysate triggers polymorphonuclear neutrophil (PMN) and monocyte activation and their subsequent generation of highly reactive oxygen species (ROS). ROS may oxidatively modify low density lipoprotein (LDL) to form oxidized LDL (Ox-LDL) which has been considered to be the key factor in the development of AS. Recent evidences showed that the level of oxidative stress already elevated in mild and moderate CRF patients, which might be caused by accumulated uremic toxins. Advanced oxidation protein products (AOPP) are one of those important macromolecule uremic toxinsfound recent years. AOPP was found to be elevated in the plasma of CRF patients by Witko-Sarsat in 1996. It was maily in albumin and characterized as having specific absorbance at 340 nm in acidic condition. Oxidation of albumin by HOC1 in vitro could get AOPP which is similar to that in plasma in CRF patients. AOPP levels in plasma have been demonstrated to be significantly elevated in mild and moderate CRF patients and increased with the deteriorate of renal function. AOPP has been ed to be a novel marker of oxidative stress in CRF as it was correlated with the other marker of oxidative stress such as malondialdehyde (MDA). In vivo, AOPP may be produced by oxidation of albumin by HOC1 formed through myeloperoxidase (MPO)-catalyzing reaction between chloride and H202 in circulating nutrophil. In vitro, AOPP has been found to be capable of inducing respiratory burst of nutrophil and monocyte, which suggested that AOPP might be inflammatory uremic toxin.Recent study showed that AOPP is in close relationship with the development of AS. AOPP level of plasma in AS patients was significantly higher than that in healthy people; thickness of carotid artery intima-media was positively correlated with the AOPP level in the plasma in hemodialysis patients; repeatedly vein injection of AOPP accelerated AS plaque formation in experimental hypercholesterolemia rabbits. These studys imply that AOPP may be involved in the development of AS and the mechanism is not clear. The aim of this study was to investigate the effect of AOPP on human endothelial cells.MethodsHuman umbilical vein endothelial cell line ECV304 was chosen as endothelial cell models to study effect of AOPP-BSA on ROS production and cell viability.1. Preparation of endotoxin free AOPP-BSAAOPP-BSA was prepared according to the method described by Witko-Sarsat et. al. Briefly, endotoxin free BSA was incubated with HOC1 at molar ratio of 1/70 and 1/140 at room temperature for 30 min and then dialyzed against PBS to remove any free HOC1 in the solution and stored at 4 癈 after sterilized by passing through a 0. 22 ( membrane. AOPP were determined by measuring absorbance at 340 nm in acidic condition and were calibrated with chloramines-T in the presence of potassium iodide.2. Measurement of ROS in endothelial cells Fluorescence microscope observation: Endothelial cells wereseeded into 24 well plate in which cover slide had been put and labeled with an oxidant-sensitive fluorescence probe, 2' ,7' -dichlorofluorescein (DCFH). The cells were then stimulated with AOPP-BSA, meanwhile PBS and BSA treated cells were used as control. The cells were fixed after 1 h incubation and observed under Nikon fluorescence macroscope and photographed.Quantitative measurement: ECV304 were first labeld with DCFH and seeded into 96 well plate at 2xl05 cells/well. The cells were then...
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