Objective:To construct and express the recombinant of human soluble TNF receptor I by E coli Methods:The cDNA coded for extracellular region of human TNFRI was amplified by RT PCR and inserted into a expression vector, pET 28a Then, the recombinant sTNFRI/pET 28a was transfected and expressed in E coli Results:After the stimulation with IPTG, sTNFRI fusion protein was effectively produced in E coli BL 21 transfected with the exogenous gene A unique band was found at 27 kD by SDS PAGE and its expression was about 31% of total protein in E coli The purified sTNFRI fusion protein was shown to be able to suppress the cytotoxicity of TNF on L929 cells and found by indirect immune fluorescence to inhibit specifically the binding of TNF with TNFR on target cells Conclusion:The recombinant human soluble TNFR I have been obtained by using the genetic engineering technology

Objective:To study the mechanisms of immune suppression mediated by Gr-1+CDllb+ myeloid derived suppressor cells (Gr-1~+CDllb~+MDSC)from tumor-bearing mice.Methods:Gr-1~+CDllb~+MDSC recruited into spleen and bone marrow of tumor-bearing mice were purified by Percoll,and suppression mediated by MDSC on T cell proliferation from spleen of naive mice was detected by flow cytometry with CSFE and FTTC-anti CD3 staining,and NO,ROS,IL-10 and TGF-β in the supematant of MDSC were detected by Griess and ELISA.Results:There were much more Gr-1~+CDllb~+MDSCs in spleen and bone marrow from tumor-bearing mouse than those of naive mouse,and suppression on T cell proliferation mediated by MDSC from tumor beating mouse was significantly increased,and there were much more NO,ROS,IL-10 and TGF-βin the supematant of these MDSC than that from naive mouse.Conclusion:MDSC from tumor-bearing mice secreted hish level of NO,ROS,IL-10 and TGF-βto induce immune suppression,and inhibite the proliferation of T cells.

OBJECTIVETo observe the dynamic expression of transmembrane (TM)-tumor necrosis factor (TNF)-alpha and secreted (S)-TNF-alpha in the development of endotoxic shock and explore the actions and mechanism of TM-TNF-alpha in liver of the rat with endotoxic shock.

METHODSEndotoxic shock in rats was induced by intravenous injection of dead gram negative bacteria E. Coli; the kinetics of TM-TNF-alpha on peritoneal macrophages and S-TNF-alpha in serum of these rats were determined. Pretreatment with TNF alpha converting enzyme antisense oligonucleotide (5 mg/kg) 30 minutes before rats were administrated dead bacteria inhibited enzymatic cleavage of TM-TNF-alpha into S-TNF-alpha. Six hours after bacteria injection, TM-TNF-alpha and S-TNF-alpha were also detected respectively. The pathological injury in the livers of rats with endotoxin shock was examined, and artery pressure was constantly measured.

RESULTSThe kinetics of TM-TNF-alpha expression in the development of endotoxic shock was different from that of S-TNF-alpha expression in serum. The expression of TM-TNF-alpha began to increase on the surface of peritoneal macrophages and liver within 30 min, after bacteria challenge and peaking within a period of 4.5 hours followed by a gradual decrease to a relatively high level which was maintained for at least 24 hours. The TACE antisense oligonucleotide pretreated rats showed remarkable increase in TM-TNF-alpha expression by peritoneal macrophages and liver (P < 0.001), and their arterial blood pressure were maintained within normal levels and there were no detectable pathological changes in their livers.

CONCLUSIONSThese findings suggested that TM-TNF-alpha may be a potent endogenous regulator involved in anti-inflammatory responses to maintain normal arterial pressure and protect liver tissue from pathological injury in during endotoxin shock. This study confirmed the important role of TNF-alpha in endotoxic shock which is not only of important theoretical significance, but also of practical interest in providing experimental basis for clinical treatment of endotoxin shock.

Animals ; Chemical and Drug Induced Liver Injury ; Disease Models, Animal ; Endotoxins ; toxicity ; Liver Diseases ; metabolism ; Membrane Proteins ; secretion ; Oligonucleotides, Antisense ; pharmacology ; Rats ; Rats, Wistar ; Shock, Septic ; metabolism ; Tumor Necrosis Factor-alpha ; secretion