| Acute hepatic failure(AHF)is refers to infection by hepatitis B virus,drug poisoning and other various reasons resulting in a large area of necrosis of liver cells in a short period of time,and thus the synthesis,detoxification,and excretion of biotransformation function obstacles or decompensation,appears in the blood coagulation mechanism of abnormal,jaundice,ascites of liver function obstacle and cause clinical syndrome.According to statistics,China's annual such patients up to 20?300000,AHF of acute onset,rapidly progressive,mortality can be as high as 80%,so the domestic and foreign scholars are actively searching for effective treatment method.Although liver transplantation is effective treatment,but with the increased incidence of liver disease,the source of donor liver is far behind the needs of patients,and liver transplantation with expensive treatment costs and other factors limit its clinical application;Bioartificial liver(BAL)can effectively improve the liver function,but because of the limitations of liver cell source and biological safety factors such as distress,its clinical application is limited;In twentieth Century 90 since the rise of the stem cell technology is gradually mature,become a research hotspot in the field of life science.Alternative therapies such as liver cell transplantation,bone marrow mesenchymal stem cells transplantation for the treatment of become a research hotspot in recent years,is expected to become a practical method for treatment of acute liver failure.Bone marrow mesenchymal stem cells(BMSCs)is a class of early self replication and differentiation potential of undifferentiated cells,which belongs to one kind of adult stem cells,has a strong plasticity,can cross the ectoderm of transdifferentiation to various tissue hepatocyte like cells,and have drawn convenient,cultured in vitro and transplantation of autologous mature technology advantages,so it is expected to become the ideal seed cells for tissue engineering and cell therapy.How to efficiently induce BMSCs into hepatocyte like cells differentiation,stem cell transplantation is the key to the treatment of liver diseases,but because of its specific differentiation mechanism is not clear,the key problem that has not been satisfactorily resolved,the clinical application is far did not achieve the desired effect.So BMSCs to explore molecular mechanisms of hepatic like cells differentiation are significant.Many experiments has confirmed that BMSCs can differentiate into hepatocyte-like but its mechanism still remains controversial.The main view of point at present are as follow:transdifferentiation theory:BMSCs are multipotential and capable of transdifferentiation to hepatocyte-like.cytomixis theory:BMSCs first mix with The host liver cells and then further differentiate into hepatocyte-like.Although definite regulation mechanism of transdifferentiation is still not clear,the majority of scholars are supportive of it since cell fusion is not found in many experiments of stem cell differentiation.BMSCS into hepatocyte like cells research content mainly divided into in vitro,in vivo studies two.In vitro differentiation research mainly focus on using different growth factor,the use of different media,and hepatic parenchymal cells or non parenchymal cells in co culture were differentiation.Cell growth factor in BMSCs differentiation process play an extremely important role,so naturally become a hot research topic.Hepatocyte growth factor has been proved to be not only able to induce liver stem cell homing to the liver cells,cells to albumin positive liver stem cell transformation can also induce the purified albumin negative hepatic stem.At the same time,the study found that hepatocyte growth factor may be through the activation of phosphoinositide 3 kinase/protein kinase B signaling pathway of BMSCs differentiate into hepatocyte like cells.Study on 'the in vivo differentiation found,purified BMSCs into hepatic progenitor cells or hepatic like cells in vivo,and can have a synthetic,parenchymal cells,liver detoxification metabolism part function.Various cytokines found in differentiation experiments such as HGF,FGF-4 etc.in physiological and pathological conditions can induce the differentiation.of BMSCs into hepatocyte like cells induced to present all the results did not permit the solid outer.In vivo experiments also confirmed that there may be some only promote BMSCs differentiate into hepatocyte like cells in the microenvironment of the liver substance;Although the discovery of clinical liver failure and liver dysfunction and other patients through autologous transplantation of bone marrow mesenchymal stem cell microenvironment specific in the liver and promote it to a functional hepatocyte like cells differentiation,so as to improve the liver function of patients,prolong the survival period,but the specific mechanism of the treatment is not clear.Yet from the"panoramic range" of BMSCs for in-depth study to the microenvironment of hepatocyte like cells differentiation.The present studies have confirmed that stem cell microenvironment directly affects and decides the direction of adult stem cell differentiation.So more and more scholars will study on micro environment as the breakthrough point and the starting point of all kinds of stem cells directional differentiation mechanism.The dry local cell differentiation of micro environment includes various cytokines,stromal cells and extracellular matrix and many other proteins,cytokines,to explore the function and mechanism of action of these substances is extremely complex,and the quantitative proteomics with a large scale,high throughput,high sensitive,and genome science,bioinformatics permeated each other,complement each other,can be used for a variety of cytokines and protein changes of extremely complex.Therefore,quantitative proteomics is a very important experiment technology of stem cell biology.Analysis of the quality and quantity of all protein it can be on the expression of a particular cell genome,can determine all the protein expression profile,relying on the efficient mass spectrometry analysis system analysis and bioinformatics,a key protein molecules can find them,and to study the interactions between biological molecules.And the research for proteomics bioinformatics development provides a convenient and effective computer analysis software,the method has the advantages of fast,intuitive.In short,at present on BMSCs into hepatocyte like cells differentiation mechanism more consistent view is:BMSCs in the induction of micro environmental factors,activation of signal transduction pathway related transcription factors through a series of selective inhibition or,thus starting the corresponding key genes,and specific transcription and translation,resulting in cell specific protein synthesis and produce the corresponding biological effects,so that the differentiated cells changes in biochemical,structural and functional.In the process of differentiation,the micro environment of certain protein plays an important role in the initiation.Therefore,contribute to the preliminary clarify BMSCs differentiating into hepatocyte like cells molecular mechanism study of protein molecular in micro environment.This topic in the establishment of acute drug rat hepatic failure model based on the differential expression at different time points of liver tissue using quantitative mass spectrometry proteomics related experimental techniques for the detection of drug-induced acute liver failure model group and the healthy control group within the micro environment of protein,and by proteomic related software and bioinformatics.Methods the differential proteins were screened from bone marrow mesenchymal stem cell differentiation may play a role of a "critical" protein,and the "critical"protein in vitro cultured BMSCs in vitro,through experiments to explore the protein induced BMSCs into hepatocyte like conversion efficiency cells,in order to deeply demonstrate the BMSCs into hepatic related mechanism of directional like cell differentiation.Chapte ? Establishment of rat acute drug-induced hepatic failure modelsObjective:Establishment of acute hepatic failure rat model by D-galactosamine plus lipopolysaccharide.Methods:69 SD rats,randomly divided into four groups:normal control group 9 rats,20 rats in group 24h,20 rats in group 48h,20 rats in group 72h.The experimental groups of SD rats by intraperitoneal injection of 0.1mg/ml D-galactosamine 700mg/kg and ?g/ml lipopolysaccharide induced acute liver failure rats,control group SD rats by intraperitoneal injection of an equal volume of 0.9%sodium chloride.Observe the general situation of four groups of SD rats,changes of 24h,48h,72h different period of rat liver function were measured,and check the corresponding period in rats liver gross and pathological changes.Result:The successful establishment of acute liver failure rats model by D-galactosamine plus lipopolysaccharide;The experimental group SD rats were administered 24 hours after appear unresponsive,slow,reducing diet,urine color yellow,beginning 48 hours suffered liver failure after obvious,SD control group rats did not appear afore-mentioned symptoms within 72 hours;Compared with the control group,the experimental group rat liver function of each time AST,TBiL,ALB,PL and NH3 showed significant change(P<0.05);with the modeling of drug actiontime prolonged,the liver function of rats gradually deteriorated.4 groups of AST,48h,72h results showed that the 24h group and the control group with significant difference(F=40.360,P=0.000);group four TBiL results shows that there is significant difference(F=61.269,P=0.000).48h group,72h group and 24h group increased significantly compared with TBiL(P<0.05),the difference was statistically significant;ALB the four groups had significant difference(F=81.336,P=0.000),48h,72h group and 24h group compared with the time prolonged,the ALB decreased significantly,with statistical significance(P<0.05).With prolonging the drug action time,liver function deteriorated sharply;four groups of experimental results of PT in fourgroups were statistically significant(F=216.335,P=0.000),24h,48h and 72h of three groupsof PT prolonged significantly,compared with the control group there was significant(P<0.05);rats in the 4 groups had significant difference(F=389.194,P=0.000),which had significant differences in 24h,48h and 72h group compared with the control group.In the liver of rat liver capsule was observed gradually tension without luster,the liver was purple,partial liver atrophy of rats,liver subcapsular large patchy hemorrhage,liver texture crisp,easy bleeding on touch,the surface of the liver showed large granular changes.At the same timewith the prolongation of time,the liver cells of rats appeared obvious hydropic degeneration,sinusoidal expansion,lymphocytes and neutrophils and other inflammatory cell infiltration;72h hpatic sinus expansion under microscope showed a mesh like,which showed congestion andhemorrhage,focal necrosis visible cell debris,serious center leaflets with zonal necrosis,partial necrosis and adjacent central or peripheral is connected with a belt,bridging necrosis,changes with acute liver failure rat liver pathology typical.Conclusions:1.This experiment adopts the D-galactosamine plus LPS was successfully established in rats with acute hepatic failure model.2.Using D-galactosamine plus LPS induced acute hepatic failure rat model is one of ideal methods to establish the animal model of acute hepatic failure.Chapter ? The two-dimensional electrophoresis of the proteins of sexperimental group and control group liver proteinsObjective:By mass spectrometric quantitative proteomics identification of 24h,48h,the difference of 72h in acute liver failure group and control group in rat liver protein expression,and by immunohistochemistry and Western blot to further verify the mass spectrometric quantitative proteomics results.Methods:According to the different time points were divided into control group,24h group,48h group and 72h group,the 4 groups will be liver tissue using iTRAQ proteome differential expression of proteins in proteomics identification of 4 technology,and through the bioinformatics analysis of these differentially expressed meaning;By immunohistochemistry and Western blot to further verify the difference identified by proteomic expression protein glutathione-S-transferase 1(MGST1)and thrombin sensitive protein 1(TSP1).Results:The four group 3106 proteins were identified by iTRAQ proteomic technology,some quantitative information of each channel protein 2820.In accordance with 2 or more peptides with high confidence(>95%),screening of differentially expressed proteins fold difference of>1.5 or<0.66 and P<0.05 standard,four groups of Chinese Communist found that the expression of the 68 proteins down regulated,73 proteins up-regulated expression.Immunohistochemistry showed that the control group liver tissue MGST1 expression was strongly positive,control group,24h,48h and 72h group,there was significant difference between the four groups(F=13.323,P=0.000).24h,48h and 72h group compared with the control group,there was significant difference(P<0.05).TSP1 there was significant difference between the four groups(F=5.375,P=0.009),48h and 72h group compared with the control group,there was significant difference(P<0.05).Western blot results showed that 24h,48h and MGST1 expression was significantly down regulated in 72h group,control group,24h,48h and 72h had statistical difference between the four groups(F=155.426,P=0.000).48h,72h group and 24h group there were significant differences(P<0.05);there was significant difference in the expression of TSP1 in the four groups(F=123.200,P=0.000).48h,72h group and control group,24h group compared with significant difference(P<0.005).verification of the two differential protein change trend and iTRAQ proteomics results are consistent.After bioinformatic analysis of differentially expressed proteins obtained galectin-3,thrombin sensitive protein 1,S100A9 may be involved in the differentiation process of bone marrow stem cells.Conclusions:1.In liver tissue of rats with acute liver failure 4 groups of samples identified 3106 proteins,and screened the difference of acute liver failure rats in different periods of protein expression in 141,including 68 down regulated proteins,73 proteins were up-regulated;2.After obtaining the galectin-3,thrombin sensitive protein 1,S100A9 of a plurality of possible with BMSCs into hepatocyte like cells induced differentiation of differential expression protein by bioinformatics analysis,might be a potential inducer of new;3.immunohistochemistry and Western blot results showed that iTRAQ proteomic expression in the liver of rats with acute liver failure identification of differential protein is a sensitive and accurate.Chapter ? Galectin-3 induced rat BMSCs to differentiate into Hepatocyte-like cellsObjective:Study on Galectin-3 on rat BMSCs differentiation into hepatocyte like cells,bone mesenchymal stem cells to provide experimental foundation in basic research and clinical application of liver diseases.Methods:Separation and purification of SD from rat BMSCs by density gradient centrifugation,and then adding 0?g/ml,0.1?g/ml,0.5?g/ml,1?g/ml,2?g/ml,Galectin-3 combined with 20ng/ml HGF,cultured in vitro induced by 28d;The morphology of cells was observed respectively at 7,14,21,28d time,by immunofluorescent staining detection of AFP,CK-18 and ALB,Q-PCR,CK-18 and ALB detection of AFP expression of mRNA gene;According to the experimental results,an additional 0.5?g/ml,Galectin-3 experimental group,induction and culture of 28d in vitro,as mentioned above,the corresponding test respectively in 7,14,21,28d.Results:Inverted microscope to a packet after induction,BMSCs form gradually transformed into hepatocyte like cells,similar to primary rat hepatic cells IAR20,wherein the 28d time period,0.5?g/ml+20ng/ml group induced culture group into IAR20 the highest proportion of similar cells;All treatment groups after induced differentiation of 7d,14d,21d and 28d immunocytochemistry staining.Results show that with the positive control group comparison,along with the prolongation of culture time,groups AFP,CK-18 and ALB fluorescence staining positive rate increased,And the positive rate in the fluorescent 28d time point 0.5 ?g/ml+20ng/ml group AFP,CK-18 and ALB;Culture of AFP,CK-18 and ALB cells in different time were detected by the detection of mRNA expression,we found that the interaction between AFP,ALB,CK-18 and mRNA group the expression of Gal-3+HGF in different concentration and induction time(F=749.082,130.386,60.470,P=0.000);with the extension of time,7d,14d 21d,AFP,CK-18 and 28d intervals and ALB mRNA expression had significant difference(F=1213.111,467.772,85.653,P=0.000),at the same time,different concentrations of Gal-3 and HGF induced bone marrow stem cells express AFP,CK-18 and ALB were significant difference(F=1349.676,133.476,242.839,P=0.000);through the analysis we found that with the prolongation of the culture time,the expression levels of AFP,CK=18 and ALB mRNA increased significantly,while the 28d expression reached the peak point in time;and the 28d g/ml Gal-3+20ng/ml HGF,0.5 induced group AFP,CK-18 and ALB mRNA expression level was the highest.At the same time,the additional 5 g/ml Galectin-3 experimental group in BMSCs after 28d culture,the experimental results appear as before the change trend.Conclusions:1.Rat BMSCs has the potential to differentiate into hepatocyte like cells;2.Gal-3+HGF,Gal-3 can induce rat BMSCs differentiate into hepatocyte like cells after differentiation,hepatocyte like cells can secrete hepatocyte specific products of AFP,ALB,CK18 etc;3.Different periods of Gal-3+HGF,the positive rate of Gal-3 BMSCS was induced to differentiate into hepatocyte like cells are not the same,with prolonging the induction time,positive conversion rate is high;4.Different concentrations of Gal-3+HGF induced BMSCs differentiation into hepatocyte like cells in different periods,the positive rate of 28d in 0.5 ?g/mlGal-3+20ng/ml HGF combined induced differentiation of BMSCS the highest;5.Separate 0.5 ?g/mlGal-3 can induce rat BMSCs into hepatocyte like cells differentiation effectively,and the secretion of hepatocyte specific products of AFP,ALB,CK18 etc. |
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