| BackgroundEndotoxin are major constituents of the outer membrane of Grarn-negtive-bacterial (GNB). Endotoxin are the most important pathogen associated molecalar patterna(PAMPs),which are not only the main causative agent of GNB infection (such as sepsis and septic shock),but also closed related to many human disease. LPS is a complex glycolipid composed of a hydrophilic polysaccharide moiety and a hydrophobic domain known as Lipid A and has to no species-specificity.The toxicity of LPS was associated with integrity structure of Lipid A. The LPS molecules are not toxic when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety,Lipid A,is exposed to immune cells,thus evoking an inflamatory response. LPS and other cell constituents are released from the bacterial cells when bacteria die or lyse. Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria resulting in the relsease of LPS.The pathogenic mechanisms of LPS are actives monocytes and macrophages to produce proinflammmatory cytokines such as tumor necrosis factor-α(TNF-α),interleukin (IL)-1,IL-6,IL-8 and IL-12. Macrophages also secrete in response to LPS,a wide variety of other biological mediators including platelet-activating factor,prostaglandin G,enzynes,and free radicals,nitric oxide. Production of these inflammatory cytokines and mediators by monocytes/macrophages contributes to the efficient control of growth and dissemination of invading pathogens.Some mechanisms have continuously been exploring although the molecular mechanisms of inflammatory response induced by LPS had been elucidated partly. So it will help us understand the mechanism of action of LPS penetrate deeply that to search for new LPS-responding molecule, and to research the function of newfound molecule. Human being lrp (hlrp) means human lipopolysaccharide responsive gene,which was obtained from differential-expression gene library of human dental pulp cell stimulated by LPS. Through PCR-based improved subtractive hybridization. The accession number in GenBank is AF143740. But the Hlrp gene sequence obtained by at that time was only part of the full-length gene,which encodes 186 amino acids of N-terminal.Before long the full-length hlrp gene cDNA sequence was also accessed into GenBank (Accession Number: NM018360). Analysis of bioinformatics demonstratd that there are 1584 base pairs in open reading frame of lrp gene cDNA sequence. The encoding sequence of lrp contains interlapping two leucine zippers and a helix-turn-helix structure.Then,full-length Hlrp gene coding sequence and rabbit anti-human Lrp antiserum were procured by Song Qin-he,one of our research group members. He subsequently carried out three respects research include intracellular location of human Hlrp, the effects of LPS on Hlrp expression in human cells But the deep research can not be carried out. For that reason, we try to learn some preliminary function information about hlrp in this study.Objective1. To evaluate the expression of Hlrp protein in normal human tissues;2. To observe Hlrp protein and quantify its relative gene expression in four cell line;3. To observe the change of proliferation and cell cycle after overexpression of hlrp in HEK293 cell;4. To observe the change of proliferation and cell cycle after RNA interference of hlrp in HEK293 cell;5. To study the differential expression of signal transduction related gene between the overexpression hlrp gene HEK293 cell and normal HEK293 cell.Methods1. Forty eight normal human tissues were collected. The expression of hlrp was detected in the tissue by microarray technique and immunohistochemistry.2. Expression analysis of full-length hlrp in HEK293, HepG2, Hela and PDC was observed by laster scanning confocal fluorescence microscope and Real time quantitive RT-PCR.3. pcDNA3.1(+)-hlrp plasmid was transfected into HEK293 cell.The Hlrp protein was detected by Western Blot.Cell proliferation after transfection was evaluated by MTT assay .The change of cell cycle was assessed by flow cylometry(FCM)4. After RNA interference of hlrp was carried out in HEK293 cell Hlrp protein was detected by Western Blot. Cell proliferation after RNA interference was evaluated by MTT assay .The change of cell cycle was assessed by flow cylometry (FCM)5. Gene chip technique was applied to study the differential expression of LPS signal transduction related gene in overexpression hlrp gene HEK293 cell.Results1. Hlrp protein was expressed in almost all tissues detected.2. Laser scanning confocal fluorescence microscope showed Hlrp protein was expressed in the cell lines (HEK293,HepG2,HeLa and PDC). Expression of hlrp was detected in HEK293 cell line and PDC cell line by real time quantitive RT-PCR.3. After transfection of pcDNA3.1(+)-hlrp,proliferation activity of HEK293 cell was significantly lower,G1 phase was blocked partly in HEK293 cell.4. Proliferation increased after RNA interference targeting against Hlrp.5. (1) Twelve differentially expressed genes related to MAPK cascade were detected,among them 6 genes were up-regulated and 6 down-regulated.(2) Thirty-eight differentially expressed genes related to MAPK signaling pathway,among them 14 genes were up-regulated and 24 down-regulated.(3) Fifty-eight differentially expressed genes related to TNF-α/NF-κB NetPath,among them 19 genes were up-regulated and 19 down-regulated.Conclusion1. Expression of Hlrp had been detected in many tissues and cell line. This result would provide data for the further study of hlrp.2. hlrp molecular overexpressed HEK293 were established,these model will obviously lay a solid foundation for further functional study. 3. Hlrp maybe a new protein which play important role in regulating cell cycle; hlrp may involve in LPS signal pathways; hlrp probably have relations with cell proliferation and cell cycle.4. Differential expression of LPS signal pathway, gene would provide data for the further study of LPS signal pathway.
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