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Interferon-inducible protein 10 (interferon inducible protein 10, IP-10), is an important class of newly discovered CXC chemokine family members. Through its specific receptor CXCR3, IP-10, combined to play an important biological functions in the body, kill microorganisms, anti-viral, anti-tumor, mediated Th1-type immune response. Endotoxin shock induced by lipopolysaccharide (lipopolysaccharide, LPS), LPS activated monocytes macrophages, endothelial cells synthesize and release various cytokines and adhesion molecules, including IP-10 as an important cytokine expression levels abnormal rise, including endotoxin shock mouse liver, lungs, kidneys, IP-10 expression were significantly elevated. IP-10 chemokine inflammatory cells reach the inflamed tissue, to some extent, to play the role of killing pathogens, but too strong an immune response and how would the body to cause damage, caused by the systemic inflammatory response syndrome, severe cases lead to the body from more than organ dysfunction syndrome. IP-10 are expressed in a variety of body tissues and cells, including the cells of the monocyte-macrophage, a variety of cells, 30 activation of fibroblasts, endothelial cells, lymphocytes, etc., and a variety of stimuli can be raised or The reduction in the IP-10 gene expression, such as interferon-γ (interferon-γ, IFN-gamma), interferon alpha (interferon-alpha, IFN-alpha), tumor necrosis factor-α (tumor necrosis factor-α, TNF-α ), interleukin-4 (interleukin, IL-4), interleukin-12 (interleukin, IL-12), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), etc.. Different stimuli in different cells of different species may induce IP-10 gene expression through different signaling pathways, regulation mechanisms are not the same. Congeners of the mouse-derived IP-10 cytokine response gene (permissive cytokine Responsive Gene-2, CRG-2) of the signal paths have been some studies, but the regulation mechanisms related to the IP-10 and its endotoxic shock expression mechanism in the model know less. The regulation of gene expression in cells of the external or internal stimulus response occurs, this is a highly complex and precise control of the process, the gene transcriptional expression regulation can copy, amplification, transcription, transcription, translation and post-translational multi-level level, but the mRNA transcription start point of the basic control of the regulation of gene expression, i.e. at a certain time, the spatial specificity based on, by a specific gene promoter and regulatory protein interactions determined. The essence of the DNA-protein / protein - protein interactions between RNA polymerase activity. In addition, it was found that the process of gene regulation, more often than not the individual factors play a role, but a variety of regulatory factors intertwined and influence each other, forming a complex regulatory network, and finally to the regulation of gene expression showed a highly ordered . In view of such a wide range of proteins and protein, protein and DNA interaction networks involved, the research strategy are endless. Among them, the promoter DNA binding protein is studied from the level of transcription regulation of gene expression, to find new proteins as a means to understand its transcriptional regulatory role for the purpose of ultimately to elucidate the pathogenesis of some diseases and gene therapy lay the foundation. Currently, the promoter DNA binding protein can be divided into two categories: first intracellular law, with its combination of protein-coding genes screened to known promoter DNA sequence, through the analysis of biological information to determine the second category of the protein; extracellular, i.e. combined in vitro with a known protein and the promoter of the recombinant DNA. Both have their own advantages, but have their own defects. Extracellular method, based on the above combination of both applications, in order to achieve both find unknown regulatory protein, but also be able to find the exact binding sites of the protein and confirmed that the two binding specificity purposes. Thus, from the point of view of \mouse liver tissue sub-binding protein interact with IP-10 start, an important regulatory elements and identified high mobility group box protein 1 (high mobility group box 1, HMGB1) verification and on further in vitro binding IP-10 gene transcription regulatory role of in-depth research. End biotin (biotin) labeled IP-10 promoter region primer, PCR amplification of the prepared IP-10 promoter region of the probe (IP-10P-Biotin). Probe were prepared endotoxin shock animal model (BALB / c mice), extract the nuclear proteins of the liver tissue with the amplification of IP-10p-biotin binding reaction, then the beads labeled streptavidin (streptavidin) separating the IP-10P-biotin-protein reactant complex, a different salt concentration buffer to elute the bound protein, eluted sample proteins using polyacrylamide gel electrophoresis separation and gel row silver staining, a comparison of endotoxin shock group and normal control group difference bands (DNA pull-down assay). The results show that, in the nucleus of liver tissue, a total of 17 proteins may be involved in the regulation of IP-10 expression in endotoxin shock mouse liver. 14 protein after LPS promoter binding and IP-10 increase after LPS suggesting that these proteins may be involved in transcriptional regulation of IP-10, while the three proteins relative to the normal control group after LPS than weakened or disappeared, and repeated the same result. With glue line cut on the results of 17 differentially identified by mass spectrometry, the resulting peptide mass fingerprinting using Mascot query software search the NCBI database, a comprehensive analysis of the five transcriptional regulatory proteins, respectively, for the high migration rate of the protein family of high mobility group box protein 1 (high mobility the group box 1 HMGB1) and high mobility group protein 2 (high mobility group box 2, HMGB2), histone (histone, H3), acetyl coenzyme A acyl aminotransferase (acetyl-Coenzyme Aacyltransferase 2, ACAA2) and thyroid hormone receptor-associated protein (thyroid hormone receptor associated proteins, TRAP). , HMGB1, HMGB2 originally discovered as nuclear factor involved in the regulation of gene transcription, mainly through modification histone bend or change the structure of chromatin / DNA and promote various protein factors in the formation of macromolecular complexes to regulate gene transcription; mainly through the acetylation level adjustment control gene transcription in the \HMGB1, the HMGB2 the acetylation involved in gene regulation; thyroid hormone receptor-related protein is a mediator of the New research coordination of transcription factor and activator of transcription, RNA polymerase II transcription factor (general transcription factors , GTFs) contact with each other so as to promote the assembly of the transcription initiation complex. These results fully identified by mass spectrometry endotoxin shock mouse IP-10 gene expression regulation may be affected by a variety of factors, and these factors are coordinated, joint action affecting the IP-10 gene expression and regulation. In view of the important role of HMGB1 in transcriptional regulation, as an important factor in the IP-10 gene regulation its extensive validation, including in vitro binding assay, immunofluorescence and Western blotting analysis in vitro IP-10 and HMGB1 binding activity of endogenous HMGB1 positioning its expression. First, the expression of the fusion protein was purified with a histidine (His) tags HMGBl their package to fluorescent microspheres coupled with nickel ions, and with the ends with biotin-labeled IP-10 promoter length to different The molar ratio of rows in vitro binding reaction, adding a fluorescence-emitting agent PE coupled streptavidin biotin, the use of liquid chip system (LiquiChip workstation) detecting the intensity of each group of DNA-protein binding. The results showed that the HMGB1 with the IP-10 promoter in vitro with high affinity, and when the DNA: protein input molar ratio of 2:1, the combination of the two tends to plateau. This result is consistent with known HMGB1 protein present on the two DNA-binding domain (HMG box domain). At the same time, we used Western blot detection of endotoxin shock HMGB1 protein expression in the mouse liver results in LPS-stimulated expression of HMGB1 in the post-nuclear proteins began to increase in the amount of 0.5 h, and subsequently returned to normal, to 3 h have increased, then began to decline, while total protein has a similar trend. In addition, we used cultured human hepatoma cell lines SMMC7721 cells observed early after LPS stimulation location test for the detection of endogenous HMGB1, HMGB1-specific localized in the nucleus, such results suggest that HMGB1 as a nuclear factor in earlier involved in the regulation of gene expression, and my room early research that showed that 1 h in LPS-stimulated endotoxin shock mouse liver tissue detected IP-10 increased and reached a peak at 3 h, thus can be concluded that the change of HMGB1 in the process of IP-10 gene expression and regulation, should occur at the transcriptional level, early HMGB1 expression increased and thus participate in the regulation of early gene upregulation of IP-10. Through the above research, we have come to the following conclusions:: biotin - streptavidin magnetic bead separation technology successfully screened to participate in the regulation of IP-10 gene transcription factor nuclear proteins, at least 17 protein may be involved in This process and the use of bio-mass spectrometry identification of protein the HMG family HMGB1 and HMGB2, histone H3 acetyltransferase, thyroid hormone receptor-associated protein;: expression in prokaryotic cells and purified with His-tagged HMGB1 protein, IP-10, and the end with biotin labeled promoter Length vitro binding to different molar ratios, using Liquichip liquid chip analysis systems in vitro validation of the interaction between the two, in the DNA: protein 2:1 when the combination of the two tends to platform. : Immunofluorescence and Western blot results showed that early HMGB1 expression in LPS-stimulated mouse liver nuclear proteins was significantly elevated, and cellular localization experiments also detect nuclear expression of HMGB1, suggesting that HMGB1 on endotoxin shock, including the role of IP-10 expression occurs early, and thus play a protective effect of early shock on the body of toxins, including IP-10. This study uses the principles of DNA-protein interactions, creating a new way of thinking from the perspective of the transcriptional regulatory genomics analysis process and its mechanisms of expression and regulation of gene transcription. Simultaneously, including the description of the emergence of the differential bands LPS in the transcriptional regulation of IP-10 occurred more complex changes, may have a variety of nucleoprotein or transcription factor involved in this process. Identification of these proteins is important for our in-depth understanding of the IP-10 gene expression regulation, but also for the clinical treatment of endotoxin shock provide a new theoretical basis and possible therapeutic target.
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