细菌内毒素损伤人内皮细胞作用机理的研究

Bacterial endotoxin is lipopolysaccharide (LPS) which presents in the outer membranes of the Gram-negative bacteria. It plays a wide range of biological functions which are highly related to the pathogenicity of the Gram-negative bacteria. Researches have been shown it is the key factor in mediating fever, inflammation, DIC, arteriosclerosis, multi-organ failure , and even shock and death The innate immunity is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs) and which can directly recognize invariant molecular structures (pathogen-associated molecular patterns [PAMPs]) which are shared by large groups of microorganisms. Recent researches showed Toll-like receptors (TLRs) are recognized as primordial receptors of innate immunity mediating activation by peptidoglycan and bacterial lipopeptides (TLR1, 2 or 6), lipopolysaccharide (LPS； TLR4), double-stranded (ds) RNA (TLR3), flagellin (TLR5), virus (TLR4, 7, 8 or 10) and unmethylated CpG DNA motifs (TLR9). Microbes can provoke, through TLRs, intracellular signaling probably involves the activation of transcription factor nuclear factor (NF)-κB in the responding cells which results in the induction of the opining of a variety of effector genes. The aim of the present study is to certify the role of TLR2 and 4 in the interaction to LPS and to scan the important adaptors in receptor-induced intracellular signaling by yeast two-hybrid system. The results showed the phagocytosis of macrophages derived from PBMC was enhanced significantly under LPS stimulation, while it was suppressed significantly when polyclonal antibodies to TLR2 or 4 were added to the reaction. The results confirmed again that TLR2 or TLR4 was a LPS receptor which mediated its signal transduction. A RT-PCR was used to amplify the full-length and their extracellular regions of TLR2 and 4 from human umbilical vein endothelial cells (hUVEC) and the results elicited that membrane receptors of hUVEC, like that of macrophges/monocytes, were also TLR2 or 4. The cDNA library of hUVEC was also constructed. With intracellular domain of TLR4 as the bait, the results from yeast two hybrid suggested that MyD88 was one of the adaptors in the pathway of TLR4 provoked intracellular signaling. These results would favor to understand the mechanisms of the interaction between LPS and the host bodies, and it is hoped to provide the novel procedures in prevention and treatment of LPS-related disorders based on these basic researches.

血管内皮细胞是内毒素作用的主要靶细胞；近来发现To11样受体4是真正介导内毒素信号的芴濉１究翁馔ü湍杆咏幌低常唇⑷似昃猜瞿谄は赴鹀DNA文库后，用含To11样受体4细胞内区域基因片段的质粒共转染酵母，筛选出内毒素信号转导过程中的接头分子。本课题将揭示内毒素的作用机制，为细菌与机体的相互作用机理提供新的认识与基础资料。