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Background: sepsis (sepsis) is still causing clinical patients developed multiple organ failure (MOF) and even death. The main mechanism is the endotoxin or lipopolysaccharide (LPS), activated monocytes - macrophage system (MPS) to release a variety of cytokines, such as the SCD14, TNF-a, IL-6, 10 and NO media caused disease. Endotoxin is a lipopolysaccharide component (LPS), while the monocyte - macrophage (MO) is a glycoprotein present on the surface - of CD14 is considered endotoxin-mediated signal transduction, activation of monocytes macrophages important receptor in LPS transmission of information from the start. Therefore, CD14 cells release inflammatory mediators related changes of CD14 expression is the basis of the body's sensitivity to endotoxin response. The monocyte-derived cell line U937 is important to study in vitro the MO cell line, most the Laboratory through VitD3 induction U937 to obtain the expression of CD14-positive cell lines, but can not guarantee the uniformity and film stability of the expression of CD14, and not conducive to endotoxin CD14-mediated signal transduction and cell release of inflammatory mediators important mechanism study, we conducted this research. Research Objective: To construct stably expressing human CD14 protein monocyte cell line U937 cell line, in order to facilitate research on the LPS-mediated CD14 expression in monocytes - macrophages (MO) film, and U937 cells CD14mRNA expression, and its mechanism of action in the secretion of cytokines. A powerful tool for the study of CD14 in LPS inflammatory response, endotoxin shock. Basic research: pDisplay/CD14 recombinant plasmid, the the PDGFR transmembrane peptide construct stable expression of a transmembrane protein CD14 molecules leukemia cell lines U937/mCD14. Methods: PCR method, CD14 was amplified at the gene level, and cloned into pGEM (R)-T Easy vector, and then transformed into DH5α bacteria, restriction enzyme digestion and purified positive recombinants sequenced Online nucleotide sequence analysis. Then pDisplay vector and PMD18-T/CD14 of single enzyme at the same time, purified CD14 and pDisplay vector using T4 ligase, ligation was carried out with restriction endonuclease Bg Ⅰ Ⅱ. Also transformed into DH5α bacteria, enzyme digestion and purified positive recombinants sequenced Online nucleotide sequence analysis. Recovery of the conventional method, the cultured U937 cells U937 cells were collected in logarithmic growth phase, the measurement test by G418 on the minimum lethal dose of U937 cells to determine the concentration of G418 selection, to recombinant plasmid pDisplay/CD14 with the Superfect transfectionreagent will transfected into U937 cells, G418 selection, CD14 protein expression detected by flow cytometry, CD14-positive cell lines U937/mCD14 initial screening. Results: CD14 gene was amplified by PCR, and successfully build the PMD18-T/CD14 plasmid, as well pDisplay/CD14 recombinant plasmid. The different concentrations of G418 medium for cell culture, and U937 cells were screened by G418 concentration of 500 ug / ml. G418 resistance using pDisplay carrier, preliminary culture transfection pDisplay/CD14 the U937 cells, flow cytometry 2 expression of CD14-positive cell lines screened U937/mCD14 (G418 screening of one month and three months group detection of CD14-PE positive cells were 23.21% and 37.01%, 2F9-FITC test results were 22.59% and 36.79%, respectively), The only part the cells found CD14mRNA and protein expression, indicating that the cell lines impure. Conclusion: Preliminary U937 cell lines expressing human CD14, provided favorable conditions for the study of CD14 in LPS inflammatory response, endotoxin shock.
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