Initial Construction Of Human CD14 Expression In Cell Line U937

Background:Sepsis(sepsis) is still causing clinical patients with multiple organ failure(MOF) and even the major cause of death.The main mechanism is endotoxin or lipopolysaccharide(LPS)-activated monocyte-macrophage system(MPS) released a variety of cytokines,if sCD14,TNF-a,IL-6,10 and NO,and other diseases caused by the media.Endotoxin is a component of lipopolysaccharide,and nuclear single-macrophage (MO) on the surface there is a sugar-protein CD14,is considered to be endotoxin-mediated signal transduction and activation of monocyte macrophages important receptor.LPS has important role in the transmission of information from start. Therefore,with the CD 14 cells related to the release of inflammatory mediators,CD14 expression is the body endotoxin-sensitive reaction.Monocyte-derived cell line U937 is the study of in vitro MO important cell lines,VitD3 through most of the research to induction of U937 was the expression of CD14-positive cell lines.However,the expression of CD14 can not guarantee uniformity and stability of the membrane,is not conducive for CD14 to endotoxin-mediated signal transduct and cell release of inflammatory mediators important mechanism,therefore,we carried out this research. Objective:This study was aimed to establish a human monocyte cell line U937 stably expressing CD14.In order to facilitate research on the LPS-mediated CD14 under a single nuclear-macrophages(MO) membrane expression,as well as in U937 cells CD14 mRNA expression,and in the secretion of cytokines in the different mechanism of action.For the Study of CD14 in the L PS inflammatory response,endotoxin shock to provide an enabling tool.Basic research:Construction of pDisplay/CD14 recombinant plasmid.To use of PDGFR-transmembrane ,in order to build a transmembrane protein in the form of stable expression of CD 14 molecules cell lines U937/mCD14.Methods:PCR method was adopted in the CD14 gene amplification level,the human CD14 gene was T-A cloned through pGEM(R)-T Easy vector,then converted to DH5αbacteria,and enzyme digested and purified of recombinant positive.To analysis on-line sequence after the sequencing.To use restriction enzymes BgⅠⅡto enzyme singly PDisplay vector and PMD18-T / CD 14 at the same time,recovered and purified CD 14 carrier and PDisplay vector to ligate both response with T4 ligase.Similarly, converted to DH5αbacteria,and enzyme digested and purified of recombinant positive. To analysis on-line sequence after the sequencing.Conventional methods of recovery of the foster U937 cells,collected U937 growing number of cells,U937 cells through G418 to the minimum lethal dose of determination tests,screened to determine the concentration of G418.U937 cells was transfected with the PDisplay/CD14 recombinant with Superfect transfection reagent.Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry(FCM). Result:PCR amplification through the CD14 gene,was successfully constructed PMD 18-T/CD14 plasmid,as well as pDisplay/CD 14 recombinant plasmid.By different concentration gradient of the G418 medium for cell culture,the U937 cells to determine the concentration of G418 screening was 500 ug / ml.To use PDisplay vector resistance for the G418,has developed a preliminary transfer pDisplay/CD14 of the U937 cell line, Flow cytometry screened 2 from the expression of CD14-positive cell lines(G418 screening on bothes group of one month and three months and CD14-PE detection of the percentage of positive cells were 23.21%and 37.01%,2F9-FITC test results were 22.59%and 36.79%),CD14mRNA found that only some of the cells and protein were expressed,indicated cell lines were impure.Conclusion:Preliminaryly established the expression of human CD14 cell line U937,for the study of CD14 in the L PS inflammatory response,endotoxin shock provided favorable conditions.