| [Background]:Besides direct toxicity to hepatocytes, Lipopolysaccharide (LPS), the ma ior component of endotoxin, also downregulate the function of phagocytosis, increased expression of monocytes/macrophage cells cytokines and inflammatory mediators. Monocytes treated with LPS showed enhance expression of TNF- a ,FasL,CD30L et al that could cause the cell death. LPS also activated the monoeytes that could increase the expression of MHC 1 antigen on T lymphocytes. All of these indicate that LPS play an important role in the pathogenesis of severe viral hepatitis B. Tumor necrosis factor relaled apoptosis inducing ligand(TRAIL), as a new member of TNF supper family, unlike TNF and Fas 1 igand, not only can induce apoptosis of cancer cells and virus infected cells, hut also normal hepatocytes.As other members of TNF super family, TRAIL belong to type II transmembrane protein. It was firstly discovered, cloned and named by Wiley in 1995 through searching the TNF isogeneic protein in the expressed sequenced tag (EST) bank. It is expressed in human T cells, natural killer(NK) cells, monocytos/macrophages, and in many other cell lines and tissues. TRAIL induces apoptosis on binding to either of two proapoptotic TRAIL receptors, TRAIL R1 (DR4) or TRAIL R2 (DR5). The complex of TRAIL and its death receptor start apoptosis by activating serious caspase molecular. Two other decov receptors, DcR1 andDcR2, express high level on normal cells surface, acted as a decoy receptor that inhibited TRAIL signaling.In this study, we firstly use RT-PCR, flow cytometry and ELISA to study the influence of LPS on monocytes to express TRAIL, and its relation to apoptosis HepG2 cell line in vitro. At the same time, wo also measured the soluble TRAIL level in the serum of patients with severe chronic hepatitis B, and compared to other liver functional indexes and LPS levels.[Objective]:To investigate the influence of LPS on monocytes to express TRAIL and its relationship to apoptosis of HepG2 cell line, and to observe the di i fcrence of soluble TRAIL level in the serum of patients wi th severe chronic hepatitis B from chronic hepatitis B and normal control.[Methods]:(1). Detecting membrane-binded TRAIL and soluble TRAIL: Use flow cytometry to measure membrane-binded TRAIL (mTRAIL). After stimulating with 0, 1, 10, 100, 1OOOng/ml LPS in 37C , 5% CO2 for 24 hours, had washed with PBS with 1% PBS for three times, monocytes were incubated with PE conjugated mouse-anti-human TRAIL antibody (0. 25 ug/l X 106 cells) in 4C for 40 minutes in the darker. After final washing with PBS/1% FBS, cells were fixed with 1% para formaldehyde - PBS and analyzed with flow cytomotry (coulter EPICS XL, Beckman). Use Enzyme-linked immunoadsorbent-assay sandwich method to measure 1 he soluble TRAIL in serum or in supernatant: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibodyspecific for TRAIL has been pre-coated onto a microplate. Standards and samples arc pipetted into the wells and any TRAIL present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal ant ibody specific for TRAIL is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the ammount of TRAIL bound in the initial step. The color development is stopped and the intensity of the color is measured in 450nm.(2). Analysis of apoptosi s: Use an Annexin V kit (BD pharMingenTM) to assay apoptosis. Apoptosis was measured by the binding of Phycoerythrin (PE)-Annexin V to the phosphatidylserine residues on the outer leaflet of the apoptotie cellular membrane. Human HepG2 cell line was cultured in 6 wells NUNC plate with DMEM medium contain 10% fetal calf serum, 5 different monocytes culture supernetant and positive controls were added into 3 parallel wells respectively. Meanwhile, 2ug/ml neutralizing monoclonal anti-TRAIL antibody was added to one parallel well...
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