Roles Of TLR4 In LPS-induced Endothelial Cell Activation And Expression Of Its Soluble Receptor

Sepsis with subsequent septic shock and multiple organ dysfunction syndrome(MODS) is the most common cause of mortality in the critically ill, including server trauma, burns, shock, major operation, etc. More and more researches have indicated that endothelial cells play a crucial role in the pathogenesis of endotoxic shock and MODS. Therefore, it is necessary to explore the activated mechanism of endothelial cells induced by LPS in sepsis and septic shock. Unlike myeloid cells such as macrophages, endothelial cells do not express membrane CD 14, and its activation induced by LPS needs LBP and soluble CD 14. Moreover, since CD 14 is not a transmembrane receptor, it lacks the ability to transfer signal into cytoplasma, which strongly indicate that there might exist the other signal molecules or transmenbrane receptors in endothelial cells. So identification of transmembrane signal molecule in endothelial cells is the pivotal research work to understand more mechanism of LPS-induced cell activation and injury. The recent discoveries and investigations on TLR4 provide a new chance to find and identify trnasmembrane receptors in endothelial cells. Several lines of evidence suggest that TLR4 is the cell surface receptor for LPS, which plays an important role in pathologenesis. Do endothelial cells express TLR4? If they do, whether it plays an important role in LPS-induced cell activaion and its signal transduction? Whether it is the transmembrane receptor for LPS in* This work was supported by the Major State Basic Research Development Program of China (No.G1999054203),National Natural Science Fund (No.30170968) and Research Fund of Medicine and Hygiene of PLA (No.0lQl05)endothelial cell or not? If TLR4 medicates LPS-induced endothelial cell activation, soluble TLR4 could be used to antagonize LPS-induced cell activation. There are still merely few scientific reports on roles of TLR4 in LPS-induced endothelial cell activation and its signal transduction. Some methods explored to study TLR4 such as gene transfection, gene mutation, and gene knock-out have some disadvantages, for example, showing low expression levels and high difficulties to do. Although antibody for TLR4 was used to study TLR4, it was muli-clonal antibody, which has low pertinence for TLR4. Therefore, it is necessary to develop monoclonal antibody of TLR4.In order to provide sufficient experimental materials, elucidate the roles of TLR4 in LPS-induced endothelial cell activation and its signal transduction, find new target and new step to interrupt endothelial cell activation induced by LPS at the membrane receptor level, and prevent and treat sepsis and septic shock, in our study, based on development monoclonal antibody for TLR4, the expression of TLR4 and the effect of LPS on the expression were observed. Meanwhile, the roles of TLR4 in LPS-induced cell activation and its signal transduction was investigated. Finally, the extracellular domain of TLR4 was cloned and its soluble receptor was expressed with pichia pastoris expression system. The main results and conclusions were summarized as follows:1.The predicated predominant B cell epitope was N-terminal No. 189-202 region peptide sequence of HKL TLR NNF DSL NV. The synthesized ploypeptide purity was 98.2% confirmed by caplilliary eletrophoresis. The peptide could induced high titer of anti-serum from 1:32 to 1:128. Meanwhile, the ployclonal antibody for TLR4 had high specificity to recognize TLR4-positive cells. These results indicate that it is practical to develop monoclonal antibody for TLR4.2. Monoclonal antibody from hybridoma B4 were obtained by cell amalgamation and ELISA screening. The culture supernatants and ascitesfluid of the hybridoma were obtained, which titers were 1:64-1:256 and 1:10 000, respectively. The antibody was IgG2, K and had high specificity and antagonistic action. It suggests that it is successful to develop monoclonal antibody.3. ECV304 cell could express TLR4.LPS could obviously upregulate the expression levels in a dose- and time-de...