The Functional Studies Of HnRNPK Regulated By RAGE

Sepsis,mediated by various factors of infection,refers to systemic inflammatory response syndrome(SIRS).It is one of the most serious complications in critically patients who are is a serious infection,major surgery,shock,severe burns and so on.Further development of the disease could result in acute respiratory distress syndrome(ARDS),septic shock and multiple organ dysfunction syndrome(MODS).Although sepsis has been taken high attention at home and abroad,which research has particularly much and the pathogenesis of sepsis has been part of the understanding,it is also lacking of effective prevention and control measures of bacterial sepsis in clinical.These facts show that the fundamental link in the pathogenesis of sepsis and its mechanism are yet to be studied in depth.Cecal ligation and puncture model(CLP)simulated appendicitis or diverticulitis perforations in the clinical,has been regarded as "gold standard" of animal models in sepsis research.Through the celiac mixed bacterial infection,this method could induce acute peritonitisis which resulted in sepsis,and it could also accurately simulate the pathophysiology that lipopolvsaccharide(LPS)continued to attack the patient who was suffering sepsis.In addition,CLP models could reflect the pathogenesis of sepsis caused by enterogenic infection well,which show that the lever of inflammatory cytokines and endotoxin in liver and lung tissue increased significantly,the inflammatory response was severe,and liver,lungs,intestines and other tissue had been badly damaged.All in all,CLP model is an ideal research experimental platform of sepsis at present.High mobility group box 1(HMGB 1),a ubiquitous,highly conserved nuclear non-histone,mainly plays a role in gene transcription regulation,the composition of nuclear protein complex in nuclear and others;when released to extracellular medium,it mediates inflammation reaction as late proinflammatory cytokines.Meanwhile,under the stimulation of HMGB 1,macrophage and single nuclear cell can secrete proinflammatory cytokines,such as TNF,and IL-1,and IL-6.Previous studies showed that Toll-like receptor-mediated signal transduction pathways was an important pathway to activate inflammatory and inflammatory reaction in early sepsis.Moreover,HMGB 1 could activate downstream signal pathway and participate in late inflammation reaction by combining with cell surface receptors,such as Toll-like receptor and advanced glycation end products(RAGE).But cell signal transduction pathway mechanism of late sepsis is not clear.The further study is required to clarify HMGB 1 and signal pathway mechanism of its receptors.What will happen in liver tissue of mice while knock out RAGE gene?And what is the molecular mechanisms?Little is currently known.These discussions could help us understand the pathogenesis of sepsis on the molecular level.It is well known that protein is the performer of the activity of life.Differential Proteomics can offer us a very good support to analysis of protein information during septic shock.In order to find out the differences between molecules,it compares the changes in the levels of protein expression between different samples,and identifies those differential proteins.The technological on Differential Proteomics research has been innovated fast,and it is possible for general research about liver proteome changes in physiological and pathological processes.Currently,there are a number of new technologies,inculding difference gel electrophoresis(DIGE)technology,the principle of which is:different samples labeled with different fluorescent dyes.It can separate more to three samples in the same piece of gel electrophoresis,as well as the internal standard is referenced in each gel.The internal standard increases the credibility of experiment,ensures that the results reflect the true biological differences,avoiding the influence of the system error.So that we can analysis the difference in protein abundance between samples strictly.Because DIGE technology is not only inherited the advantages of two-dimensional gel electrophoresis,but also high throughput and high dynamic range.It also improves the resolution.Currently,quantitative proteomics research has become one of the most commonly methods.Based on 2-DE,through marking different protein samples with different fluorescent dyes,DIGE separates more to three different protein samples in a two-way gel glue,in the while each gel introduces into the same marker,which increases experiment of credibility,avoiding system errors on experiment results,and ensuring the real reliable of results.DIGE is a method that fluorescent dye labels protein samples before the 2-D electrophoresis.The biggest advantage is the integration of multiple markers and dyes DeCyder 2D analysis software.DeCyder 2D analysis software utilizes the total point detection algorithm,able to automatically detect the fluorescence image,background removal,quantitative,normalized and gel matching.In addition,it avoids the influence of anthropogenic factors eliminating the errors of different operators.In order to those advantages,using this technology platform will have a better opportunity to reveal differences proteome mapping in liver tissue of mice in septic shock,providing a new methods about the molecular mechanisms of sepsis for future study.Several C57/BL6 mice of Wild type and RAGE knock-out were taken,respectively,made control(sham)and sepsis model(CLP),divided into WT-sham,W T-CLP,KO-sham and KO-CLP four.After 8 h bleed mice through cervical,then extract the liver protein,and use 2D-DIGE technology platform of proteomics,and separate the four groups of mice liver tissue proteome.Then identify the different protein by MALDI TOF/TOF,analysising these datas,wo found more than 20 different proteins.As papers reports,hnRNPK was found highly expressed in tumor tissues,and studies have confirmed its expression in tumor cells such as pancreatic cancer,lung cancer,prostate cancer and so on;When a chronic myelogenous leukemia vary rapidly,hnRNPK increase.Its works through regulating gene expression related to cell proliferation and influence the occurrence of tumor development.It shows,including hnRNPK,The mechanism of hnRNP family members on development and transfering of the specific is worth studying in-depth,in a variety of tumor occurrence.Chronic inflammation and oxidative stress,are closely associated with the formation and development of various diseases.The research about hnRNPK is less in inflammation,which ultimately determine the heterogeneousnuelear ribonucleoprotein K,hnRNPK,for interested target protein,further functions discussed in this paper.Taking four wild type rats and four knock-out RAGE gene type mice,each group randomly was divided into control group and CLP model group.All control group was preformed fake surgery(Sham),experiment group was preformed CLP model model,constituency respectively for W-Sham,and W-CLP,and KO-Sham and KO-CLP.Each group was executed small rats after 8 h,which livers were taked for following experiment.extraction of liver protein,then using proteomics 2D-DIGE technology platform,the four groups of mice liver tissue proteome separation,using MALDI TOF/TOF difference techniques of biological mass spectrometry identification of protein,through data analysis,screening by more than 20 different proteins,literature reports,hnRNPK was found in many are highly expressed in tumor tissues,studies have confirmed its in pancreatic cancer,lung cancer,prostate cancer and other tumor cells and tissues in expression;In chronic myelogenous leukemia entered rapidly varied period,hnRNPK expression will increase.Its main through regulating gene expression related to cell proliferation and influence the occurrence of tumor development.This shows,including hnRNPK hnRNP family members in a variety of tumor occurrence,development and transfer of the specific mechanism is worthy of in-depth study,chronic inflammation and oxidative stress,such as closely associated with the formation and development of various diseases,research on hnRNPK less in inflammation,which ultimately determine the nuclear inequality within a ribonucleoprotein K(heterogeneousnuelear ribonucleoprotein K,hnRNPK)for interested target protein,further functions discussed in this paper.Protein is the most main function exerciser,and each physiological activity needs many protein moleculars participate in and cooperate together.Through the mutual role of covalent bonds,salt key,and sparse water,various protein members gather into a big unit to complete a species physiological activity.Usually such a large collection of protein molecules referred to as "multi-protein complexes"(MPCs).In various physiological and pathological conditions,protein expression,with the change of the level of protein expression,subcellular organelles position and modifier state,the composition of protein complex which plays a role in variety of physiological functions is always in dynamic changes,and these changes could directly explain cellular response mechanisms in various conditions.Therefore,the study of the MPCs composition and dynamics will contribute to a better understanding of the molecular mechanisms of cell biology functional development.During the Preparatory Work,we spilted the four groups of samples by the DIGE technology,hnRNPK is a up-regulation protein in W-CLP/W-Sham,a down-regulation protein in KO-CLP/KO-Sham.However whether hnRNPK is consistent with DIGE results changes in protein and RNA levels,we donnot know;And how liver injury groups before and after sepsis in mice;where is the HnRNPK distribution in mice liver cells as well as the positioning of the inside cells,and what is the protein complex forms in sepsis?This paper conducted the following experiment to solve these problems,first using HE staining method,observation of liver injury of wild type and RAGE knock out mice with different time CLP model groups;detect its expression in protein and RNA levels of mice liver,validation wether they are consistent with the results of 2D-DIGE;And how liver injury groups before and after sepsis in mice;Giving the AGE at different time points,observate the complex aggregation and shift condition of hnRNPK;Finally the method of using BN-PAGE combined mass spectrometry,isolation,identification hnRNPK related protein complex composition of the protein.Throughout the experiment plan and result,we can draw the following conclusion:1.Through HE staining we find that liver is badly damaged in sepsis mice;2.The validation in protein and mRNA levels agree with DIGE level validation results;3.Immunohistochemistry IHC test result suggests that hnRNPK is distributed in the nucleus and cytoplasm,but mainly in the nucleus;4.Cell immunofluorescence results showed that hnRNPK are expressed all in cytoplasm and nucleus in Raw264.7 cells and HepG2 cell,but it is mainly distributed in the nucleus;there is not apparent shift phenomenon in hnRNPK complex after the stimulus of AGE;5.The study extract soluble protein complexes from liver tissue of mice and find differential protein complexes,and identify the ingredients of differential protein complexes.By DIGE separation technology and MALDI TOF/TOF mass spectrometry identification technology combined with the use of the genes in mice,the method of can provide us with a method of looking for target protein.