| Lipopolysaccharides (LPS) constitute the major cell wall component in all Gram-negative bacterial (GNB) families. It is now believed to be the primary trigger key of pathophysiologic events in sepsis. GNB sepsis or septic shock, is a catastrophic and frequently fatal systemic syndrome characterized by hypotension, inadequate organ perfusion and multiple organ failure. It cause an estimated a mortality rate of 10-15% in children and up to 40% in adults.lt is previously recognized that release of early proinflammatory cytokines such as tumor necrosis factor-a and interleukin-1 can mediate the development of systemic inflammatory response and subsequent tissue injury, human high mobility group-1 protein(HMGBl),a ubiquitous DNA-binding protein ,was recently identified as a potential late mediator of delayed endotoxin lethality.The discovery of a cytokine activity for HMGB1 and studing the significance of HMGB1 in sepsis might be of importance to contribute to understanding of sepsis with subsequent multiple organ dysfunction(MODS) and guiding the utilization of innovative therapies. So, in our studiesinclude following three parts:1. Cloning and expression of the human high mobility group-1 protein gene in E.coli: Human HMGB1 gene was amplified by RT-PCR from human primary peripheral blood mononuclear cells and cloned into vector pGEM-T easy. After sequenced,it was subcloned into prokaryotic expressive vector pGEX-4T-2 . Expressed recombinant plasmid pGEX-HMGl was constructed. Having induced by EPTG for 4 hours,the expression of recombinant Mr 56kD fusion protein GST-HMG1 was confirmed by SDS-PAGE. The expressed fussion protein was about 23% of total bacterial protein by gel thin-layer chromatography-scanning.2. Purification and Identification of recombinant human HMGB1:HMGB1 was eluted from GSTrap FF affinity chromatography after thrombincleavage. The Mr 30kD purified protein was obtained . To examined whetherpurified protein HMGB1 might stimulate TNFa secretion in human monocyteculture ,it was co-cultured with THP1 cells . THP1 cells were stimulated withrecombinant human HMGB1 in 0,0.1礸/ml,1.0礸/ml,10礸/ml for 6 hours. Theability of HMGBl's inducing TNF-a release in THP1 cells was observed byELISA. We found that the purified protein HMGB1 significantly caused theincreasing release of TNF-a from THP1 cells. Then it was injected into Balb/crats through tail vein at a dose of 5mg/kg.Eight hours later,the rats were killed toobserve the inflammatory response in their livers and lungs.3. A study on the rule of TNF-a secretion in human THP1 induced by HMGB1: In order to study the rule of TNF-a secretion in human THP1 induced by HMGB1, THP1 cells were stimulated with HMGB1 in O.l礸/ml,1.0礸/ml,10礸/ml,20礸/ml,50礸/ml for 6 hours. In another experiment, THP1 cells were stimulated with 20礸/ml HMGB1 and 20礸/ml HMGB1 mixed lOOng/ml LPS for 0,2h,4h,6h,8h,10h,12h,24h respectively. The ability of HMGBl's inducing TNF-cc release in THP1 cells was observed by ELISA. There were two release peaks of TNF-a from THP1 cells after induced by the HMGB1 alone and mixed group, in 0-24 hours. We found that HMGB1 markedly caused the release of TNF-a in a dose-dependent way(from 0.lg/ml to 50g/ml) and raised the level of apoptosis.Above all,these experiments indicated that HMGB1 could markedly induce apoptosis and TNFa secretion from THP1 cells.Taken together,these results suggest that HMGB1 play a important role during inflammatory process. It may be of great significance to study on the biological functions of human HMGB1 and the preparation of some monoclonal antibody against human HMGB1.
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