| The liver is one of the most important and the most complex organs in human body.Various factors such as drugs,alcohol and virus can cause liver damage,the resulting hepatitis,fatty liver disease and cirrhosis of the liver are common liver disease in our country.The further developmental irreversible liver failure becomes one of the reasons for the higher mortality rate of the patients with hepatic injury.But most routine liver treatment effect is not ideal.Stem cells were a group of cells having self-renewal and multi-directional differentiation potential.According to its different sources,stem cells divided into embryonic stem cells and adult stem cells.Umbilical cord mesenchymal stem cells(UCMSCs)were found in Wharton's jelly surrounding tissue of umbilical cord blood vessels,and had properties of adult stem cells derived from mesoderm.Relative to other sources such as bone marrow,cord blood,fat of MSCs,UCMSCs had many advantages,including of convenient acquisition,differentiation potential,rich source,proliferation ability,low rejection immunogenicity.It can avoid the pains of patients using autologous stell cells transplantation and have no quantity restrictions of autologous stell cells.Because of unique immune regulation,self-renewal and cross layer multi-directional differentiation potential,etc.,UCMSCs were the ideal seed cells for the treatment.Stem cell transplantation therapy as a complementary and alternative treatment for orthotopic liver transplantation,it provided a new train of thought and method for a variety of end-stage liver disease treatment.It was found in animal experiments and clinical studies that MSCs had good therapeutic effect on liver fibrosis,acute liver failure and drug-induced liver injury.However,the mechanism had not been fully elucidated,the besttransplantation way of stem cells and the best therapy protocol were not clear,the clinical efficacy and safety for a long time was need to be further confirmed.These factors became the main factors restricting the wide use of the stem cells.The present study found that MSCs may play role through a variety of ways,the exact mechanism remains controversial,mainly had the following two hypotheses:(1)supplementary effect,MSCs can directionally differentiate into liver cells instead of the necrotic cells;(2)immunomodulatory effect,it can stimulate and repair the original liver cell.Both roles may complement each other and made therapeutic action together.Immune regulation was inherent characteristics of stem cells,and differentiation of stem cells affected by many factors.If the key factor influencing the stem cell directional differentiation can be found,it will provide great help for the clinical application of stem cells.In order to better study the differentiation of stem cells,the researchers set up different induced method in vitro.Usually stem cells were induced by various cytokines of different concentrations,and induction time was among 8to 10 weeks.These methods had disadvantages of long induction time and low induced efficiency.At present,it had shown that surrounding microenvironment of stem cells caused by other cells and humoral played important roles on the proliferation and differentiation of stem cells.When the microenvironment changed,stem cells can be activated to proliferate or differentiate into different function cells.Differentiation of stem cells involved many changes of a variety of receptor expression and downstream signaling molecules pathway(e.g.,ERK,Wnt,Notch,MAPK,m TOR signaling pathways,JAK).Different signaling pathways started different differentiation.Study found UCMSCs can differentiate into various tissues and cells in vitro,and transcription factors associated with liver cells development and hepatic progenitor/stem cell markers expressed highly in UCMSCs,which indicated UCMSCs may have more advantages in the differentiation into liver cell.Studies had reported that one of liver-enriched transcription factors-hepatocyte nuclear factor-4?(HNF-4?)played an important role in theformation of liver cells from embryonic cells.Previous researches suggested HNF-4? may play an important role in the process of directional differentiation of stem cells into hepatic cells.It had reported that HNF-4?was a new target gene of AMPK,so further researches on the effect of HNF-4? in the directional differentiation of UCMSCs into liver cells will help to clarify the molecular mechanism of directional differentiation of UCMSCs into liver cells.Part one Study of umbilical cord mesenchymal stem cells for the treatment of liver injury and directional differentiation in vivoObjective:To establish models of acute liver injury and liver fibrosis in rats,to observe and evaluate the therapeutic effect of UCMSCs in two kinds of liver injuried model rats,to study migration of UCMSCs after intravenously transplantion,and the differentiation of stem cells settled in the liver tissue.Methods:1 The full-term fetal umbilical cord was collected under aseptic condition,and primary cell was cultivated by tissue adherent method.The immune phenotype of UCMSCs was identificated by flow cytometry,and then differentiation potential of UCMSC was identified in vitro.Fluorescent dye PKH26 was used to mark UCMSCs.The rat model of acute liver injury was made by a single abdominal cavity injection of 50% CCl4;Rat liver fibrosis model was prepared by intragastric administration of TAA for 6 weeks.2 Group of experiments1)Groups of acute liver injury experiment90 rats were randomly divided into 5 groups: normal control group;model group;vehicle group(the rats were injected with 500 ?L physiological saline through the tail vein after CCl4 treatment);the treatment group(the rats were injected with suspension of 2 × 106/m L stem cell through the tail vein after CCl4 treatment);treatment control group(the normal rats were injected with suspension of 2 × 106/m L stem cell through the tail vein).Each group was randomly divided into three time points: 3h,2d,7d,and 6 rats in eachtime points.The rats were sacrificed at the designed time point after treatment,and the specimens were collected.2)Groups of liver fibrosis experiment120 rats were randomly divided into 5 groups: normal control group;vehicle group;single treatment group;four treatments group and normal control group.The therapeutic method of single treatment group was transplantation 1 × 106 UCMSCs through rat caudal vein at a week after the last time to give TAA.The rats were sacrificed at 3 h,2 w and 4 w after transplantation respectively,at the same time specimens were collected.The rats of four treatments group were transplantation 1 × 106 UCMSCs through rat caudal vein at a week after the last time to give TAA,once a week and for4 w.The rats were sacrificed at 1 w and 1 y after the last transplantation.The rats of vehicle group were injected the same volume of physiological saline.The rats of normal control group did not make any processing.The rats of treatment control group were injected 1 × 106 UCMSCs by tail vein.6 rats in each time point were sacrificed to collect specimens at the same time points of treatment group.3 Evaluation indicatorsThe serum levels of ALT,AST,MDA and TBIL were used to evaluate the liver function of acute liver injury model rats after UCMSCs transplantation.The serum concentrations of BAM,TBIL,AKP,ALB and GLB were used to evaluate liver function of liver fibrosis model rat after UCMSCs transplantation.HE staining and Masson staining were used to observe the effects of UCMSCs transplantation on liver tissue pathology structure.The frozen section of liver,heart,spleen,lung,kidney,and brain tissue were observed to track migration of UCMSCs after transplantation by fluorescence microscope.Immune histochemical method was used to observe UCMSCs directional differentiation in rat liver.Results:1 Isolation,culture and identification of UCMSCsOur laboratory had been successfully makes stem cells from umbilicalcord.Cell proliferation ability had no obvious change after extending 10 times.These cells high expressed mesenchymal cells signs and no expression of hematopoietic cells and endothelial cells symbols and major histocompatibility antigens.Thees cells had differentiation potential with osteogenesis and adipogenic.2 Effect of fluorescence labeling on UCMSCsThe morphology of UCMSCs did not change weather labled by PKH26 or not.They were long fusiform,swirl or radial spread geometry under inverted microscope.Dye was distributed evenly in the cell membrane.Red fluorescence was found under the fluorescent microscope.The cell outlines was clear,and the mark rate reached 100%.From the third generation UCMSC,they were labeled by PKH26,then along five passages,each generation cultured five days,cell fluorescence intensity gradually weakened,but it still can be detected at the fourth passages.The absorbance value of two groups of cells in each point time also had no statistical significance(P >0.05).This suggested that the fluorescent dye had no effect on cell proliferation.3 The distribution of UCMSCs in major organs of rat body after transplantationA large number of red fluorescence cells were visible in rat liver tissue of treatment group under the fluorescent microscopy.Fluorescence labeled UCMSCs were also visible in the liver tissue of treatment control group rats,but the quantity compared with treatment group significantly reduced(P <0.05).Compared to treatment control group,the number of fluorescent cells in spleen and lung of treatment group significantly reduced(P < 0.05).The fluorescent cells of two groups were occasionally visible in heart,kidney,and brain tissue.4 The distribution of UCMSCs in the liver after transplantationA large number of red fluorescence cells were visible in rat liver tissue under the fluorescent microscopy.UCMSCs mainly distributed around the portal area of liver tissue and blood vessels.Three fields were randomlyselected to camera and analysis.Compared with that of the acute liver injury group,the number of UCMSCs in liver tissue of normal control group reduced significantly(P < 0.05).Compared with positive cells marked PKH26,positive cells of phagocytes markers EMR1 significantly reduced(P < 0.05).The results showed that 86.32%-99.76% of PKH26 positive cells were not EMR1 positive,which suggested that these cells were not devoured by phagocytes.5 Effect of UCMSCs transplantation on rat liver function1)Effect of UCMSCs transplantation on liver function of acute liver injuried ratsCompared with those of normal control group,serum levels of AST,ALT,MDA and TBIL in model group increased significantly at 24 h(P < 0.01).It suggested model was build successfully.Compared with those of normal control group,serum levels of AST,ALT,MDA and TBIL elevated significantly in model group and vehicle group after UCMSCs transplantation3 h,2 d and 7 d,(P < 0.05 or P < 0.01);Compared with those of vehicle group,serum levels of AST,ALT,MDA and TBIL in treatment group decreased significantly(P < 0.05);Compared with normal control group,there was no significant differences of MDA and TBIL levels in treatment 2d group(P >0.05),levels of AST and ALT had no significant differences in treatment 7d group(P > 0.05).2)Influences of transplanted UCMSCs on the body weight and liver function of liver fibrosis ratsIn the process of madding rat liver fibrosis model,rat weight growed slower in model group,compared with the control group.After UCMSCs treatment,compared with those of normal control group,weights were both lower significantly in model group and treatment group(P < 0.05).Compared with those of model group,the weights were higher significantly in 4w and 4t groups(P < 0.05).In addition to the 4w and 4t groups,compared with the previous points of time,body weights increased significantly in the rest of the groups(P < 0.05).Compared with those of control group,the serum concentrations of BAM,TBIL and AKP were higher significantly and ALB/GLB ratio reduced significantly in vehicle 3h,2w,4w and 4t groups,BAM increased significantly in vehicle 1y group(P < 0.05).Compared with those of vehicle group,the level of BAM reduced significantly in treatment 3h,2w,4w,4t and 1y groups(P < 0.05),the levels of TBIL and AKP reduced significantly in treatment 2w,4w and 4t groups(P < 0.05),ALB/GLB ratio increased significantly in treatment 3h,2w,4w,4t and 1y groups(P < 0.05).6 Effect of transplanted UCMSCs on liver pathological structure of liver injuried model rats.1)Effect of transplanted UCMSCs on liver pathological structure of acute liver injuried model ratsIn the control group,normal liver cells can be found in rat liver slices by routine HE staining,and the structure of hepatic lobule was complete,liver plate were arranged a funicular radioactive sample round with central vein as the center.It was visible inflammatory cells,liver cells degeneration and necrosis in liver tissue of model group.Compared with model group,the average visual field numbers of inflammatory cells decreased significantly after transplantation UCMSCs in 3h,2d and 7d groups(P < 0.05).Compared with model group,the average vision necrotic cells decreased significantly in2 d and 7d group after transplantation UCMSCs(P < 0.05).2)Effect of UCMSCs therapy on pathological structure of liver fibrosis ratsIn model group,it was visible fibrous tissue hyperplasia,liver lobule structure destruction.And liver cell regeneration formed pseudolobule.Inflammatory cells infiltration and bile duct cell hyperplasia can be found.Hyperplasia of the fiber can be dyed blue by masson staining.Liver fibrosis reduced significantly after transplantation UCMSCs,with the extension of treatment time,the degree of liver fibrosis gradually reduce.Bile duct cells severe hyperplasia and a large number of heterocyst can be seen in model group after 1 year,but the treatment 1y group not be seen.Hyeing resultsanalysis showed that degree of liver fibrosis reduced significantly in treatment2 w,4w,4t and 1y groups,and fibrosis degree in the treatment 4t and 1y groups were lighter than 3h,2w and 4w groups compared with vehicle group(P < 0.05).7 Differentiation of UCMSCs into hepatocyte-like cells in liver tissue1)Differentiation of UCMSCs into hepatocyte-like cells in CCl4-damaged liver tissueIt can be detected a small number of liver specific markers in liver tissue,but not see hepatocyte-like cells,according to the results of immunohistochemical,after UCMSCs transplantation 3 h.The hepatocyte-like cells were visible and they expressed liver cell markers of AFP,CK18,ALB and TPH2 after UCMSCs transplantation 2 d and 7 d in live tissue.Compared with vehicle group,liver cell markers of each group increased significantly(P< 0.05).Compared with those of 3h group,the expression quantity of AFP,ALB and TPH2 increased significantly in 2d and 7d group(P < 0.05).Compared with those of 2d group,the expression of CK18,AFP,ALB and TPH2 increased significantly in 7d group(P < 0.05).2)Differentiation of UCMSCs into hepatocyte-like cells in TAA-damaged liver tissueA small number of liver specific markers can be detected in liver tissue,but no hepatocyte-like cells,according to the results of immunohistochemical,after UCMSCs transplantation 3 h.The hepatocyte-like cells were visible and they expressed liver cell markers of CK18,ALB and CYP3A4 after UCMSCs transplantation 2 w in live tissue.Compared with vehicle group,liver cell markers of each group increased significantly(P < 0.05).Compared with those of 3h group,the expressions of CK18,ALB and CYP3A4 increased significantly in 2w,4w,4t and 1y groups(P < 0.05).Compared with those of 2w group,the expressions of ALB and CYP3A4 increased significantly in 4w,4t and 1y groups(P < 0.05).Compared with those of 4w group,the expressions of CYP3A4 and ALB increased significantly in 4t and 1y groups(P < 0.05).Compared with those of 4t group,the expressions of CK18,ALB,and CYP3A4 increased significantly in 1 y group(P < 0.05).Conclusions:1 Transplant action of UCMSCs could improve liver function and liver tissue pathology structure of acute liver injuried and liver fibrosis model rats.2 Transvenously transplanted UCMSCs could migrate and settle in the damaged liver tissue;and directionally differentiate into hepatocytes with specific markers in the liver tissue.This might be one acted pathway of UCMSCs to liver injury.Part two Experimental research of directional differentiation of umbilical cord mesenchymal stem cells into hepatocytes in vitroObjective: To establish and optimize the method of directional induction of mesenchymal stem cells differentiatiing into hepatocytes in vitro,and to measure the characteristics and functions of the induced cells after differentiation.Methods:1 The livers of normal male SD rats and acute liver injury model rats were perfused,and then manual homogenated,to prepare the liver homogenate supernatant extracts(LHS)and CCl4-LHS.The application concentration of LHS was 100 mg/m L,and that of CCl4-LHS was 50 mg/m L.2 GroupsThe third-generation UCMSCs of the density of 1.2 × 105 cell a well were inculated in 25 cm2 culture bottles for 1-2 days.The inductive medium was joined into culture bottle after cells grown to 80% confluence.Cells was divided into eight groups,incluing LHS control group,LHS-3d group,LHS-5d group,LHS-7d group,CCl4-LHS control group,CCl4-LHS-3d group,CCl4-LHS-5d group and CCl4-LHS-7d group.Cells and cell supernatant were collected after treated 3 d,5 d and 7 d.3 The levels of protein and gene expression of AFP,CK18,ALB and CYP450 enzyme after induction were determinated by using western blot technology and real time PCR technology.4 The metabolic activities of CYP450 enzyme system CYP1A2,CYP2A6,CYP2C9,CYP2C19,CYP2E1 and CYP3A4 were evaluated by HPLC/MS/MS.Results:1 Effects of LHS and CCl4-LHS on cell morphology in vitroUCMSCs were observed under inverted microscope.The cells of control group were long spindle cells of fiber samples.They were spiral arranged.UCMSCs gradually changed into heterocyst-like cells after LHS induction 3 d.Most of the cells became irregular shape or circular after 7 d induction.UCMSCs began to shrink after CCl4-LHS induction 1 d.The cells were circular or elliptic after 3 d induction.The cell morphology not changed at 5 d and 7 d.2 Effects of LHS and CCl4-LHS on hepatocytes related protein expression of UCMSCs in vitroWestern blot results showed that compared with those of control group,the expressions levels of AFP,CK18,ALB,CYP1A1/2,CYP2A6,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4 were enhanced significantly after LHS and CCl4-LHS induced at different time points(P < 0.05).Among them in LHS group,the AFP expression quantity was peak at d 5,and then decreased.The expressions of CK18 and ALB increased with the extension of induction time.CYP450 enzyme family protein expressed quantity was biggest at d 5 and d 7(P < 0.05).In CCl4-LHS induced group,the AFP expression quantity was peak at 3 d after induction,and then decreased.The expression of ALB increased with the extension of induction time.The expressions of CK18 and CYP450 enzyme family proteins were most at d 3(P < 0.05).Compared with LHS-3d group,six indexes expression levels increased and four indexes expression levels reduced significantly in CCl4-LHS-3d group;Compared with LHS-5d group,two indexes expression levels increased and eight indexes expression levels reduced significantly in CCl4-LHS-5d group;Compared with LHS-7d group,eight indexes expression levels reduced significantly in CCl4-LHS-7d group(P < 0.05).3 Effects of LHS and CCl4-LHS on hepatocytes related gene expression of UCMSCs in vitroReal time-PCR results showed that compared with those of control groups,the expression levels of AFP,CK18,ALB,CYP1A1/2,CYP2A6,CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4 were enhanced significantly in different time groups of LHS and CCl4-LHS(P < 0.05).Among them in LHS groups,expression level of AFP peaked during induction of d 3,the expression levels of ALB and CK18 increased with the extension of induction time,the gene expression levels of CYP2C9,CYP2D6,and CYP3A4 were peaked at d 5,the gene expressions levels of CYP1A1/2,CYP2A6,CYP 2C19 and CYP2E1 were maximum amount at d 7(P < 0.05).In CCl4-LHS groups,the expression levels of AFP peaked during induction at d 3,and then decreased.The expression levels of CK18 and ALB were increased with the extension of induction time.The expression amounts of CYP450 enzyme family of CYP1A1/2 and CYP2A6 were maximum amount at d 5.The expression level of CYP2C9 was most at d 3,and the largest expression amounts of CYP2C19,CYP2D6,CYP2E1 and CYP3A4 peaked at d 7(P < 0.05).Compared with LHS-3d group,nine indexes expression levels increased and one indexe expression level reduced significantly in CCl4-LHS-3d group;Compared with LHS-5d group,four indexes expression levels increased and five indexes expression levels reduced significantly in CCl4-LHS-5d group;Compared with LHS-7d group,three indexes expression levels increased and six indexes expression levels reduced significantly in CCl4-LHS-7d group(P< 0.05).4 Effects of LHS and CCl4-LHS on the metabolic activity of CYP450 enzyme family of UCMSCs in vitroAfter add six kinds of CYP450 enzyme substrate metabolism,their metabolites were detected.They were 1,7-dimethyl xanthine,7-hydroxyl coumarin,4-hydroxy tolbutamide,6-hydroxy chlorzoxazone,1-hydroxy midazolam respectively.It explained that there were activities of CYP1A2,CYP2A6,CYP2C9,CYP2E1 and CYP3A4 enzyme in the induced cells.After protein quantitative,results showed that,compared with control group,each group of enzyme products increased significantly(P < 0.05).The CYP1A2 enzyme activity was the highest in the LHS-5d group,followed by CCl4-LHS-3d group,CCl4-LHS-5d,7d group gradually reduced(P < 0.05).CYP2A6 enzyme activity was the highest in LHS-5d,CCl4-LHS-3d,CCl4-LHS-5d group(P < 0.05).There were no significant differences between groups of CYP2C9 enzyme activity(P > 0.05).CYP2E1 enzyme activity was the highest in LHS-3d and LHS-5d group(P < 0.05).CYP3A4 enzyme activity was the highest in the LHS-3d group(P < 0.05).Compared with those of LHS-3d group,three indexes expression levels increased and one indexe expression level reduced significantly in CCl4-LHS-3d group;Compared with those of LHS-5d group,three indexes expression levels increased significantly and two indexes expression levels were the same in CCl4-LHS-5d group;Compared with LHS-7d group,one indexe expression level increased significantly and four indexes expression levels were the same in CCl4-LHS-7d group(P < 0.05).5 Effects of LHS and CCl4-LHS on synthesis and secretion albumin and urea of UCMSCs in vitroCompared with those of control group,ALB secretions were significantly higher in LHS groups,and secretions increased along with the induction time(P < 0.05).Compared with those of control group,the secretion of urea were significantly higher in LHS groups,and production increased along with the induction time(P < 0.05).Compared with those of control group,the secretion of ALB were higher significantly in CCl4-LHS groups,and the production was the largest in LHS-3d group,then gradually reduced(P < 0.05);Compared with those of control group,the secretion of urea were significantly higher in CCl4-LHS groups,the production was the largest in LHS-3d group after reduced(P <0.05).Compared with those of LHS-3d group,ALB and urea levels were highersignificantly in CCl4-LHS-3d group;Compared with those of LHS-5d group,ALB levels were higher and urea levels were lower significantly in CCl4-LHS-5d group;Compared with those of LHS-7d group,ALB levels were same and urea levels were lower significantly in CCl4-LHS-7d group(P< 0.05).Conclusions:1 In vitro LHS and CCl4-LHS could induce UCMSCs differentiate into hepatocytes,which expressed hepatocellular proteins and genes highly,had CYP450 enzyme activity,secreted albumin and urea.This showed normal liver tissue and injuried liver microenvironment could induce the directionally differentiated of UCMSCs into hepatocytes,which could partially perform the hepatocytic function.That might be the mechanism of UCMSCs improving liver function.2 In vitro compared with LHS,CCl4-LHS had higher efficiency when inducing UCMSCs.Because the induced concentration of CCl4-LHS was lower,and the induction time was shorter,but the induced effect were similar.It prompted that liver injury microenvironment was more advantageous to differentiate UCMSCs into hepatocytes.Part three Effect of LKB1/AMPK/HNF-4? signaling pathways on the directional differentiation into hepatocytes of umbilical cord mesenchymal stem cellsObjective: To study the role of LKB1/AMPK/HNF-4? signaling pathways in UCMSCs directional differentiation into hepatocytes.Methods:1 The changed genes were found by gene chip experiment after UCMSCs differentiated into hepatocytes,and upstream related signaling pathway of the key gene were looked for by IPA analysis.2 GroupsGene chip experiments were divided into two groups: control group and LHS group: C-1,C-2,C-3,LHS-1,LHS-2,LHS-3,and each group had three samples respectively.Signaling pathways experiment was divided into 6 groups including control group,CCl4-LHS-3d group,AMPK activation agent AICAR group(1m M),AMPK inhibitors Dorsomorphin 2 HCl(DM)group(10?M),CCl4-LHS+AICAR group and CCl4-LHS+DM group.3 The protein and gene levels of LKB1,AMPK and HNF-4? were detected by Western blot technology and real time-PCR technology after induction.The effects of HNF-4? inhibitors-BI6015 were observed by real time-PCR technology on differentiation.The effects of AMPK activator AICAR and antagonist Dorsomorphin 2 HCl on differentiation were observed by using western blot technique.Results:1 Quality of chip dataSignal Histogram,Relative Signal Box Plot,Pearson's Correlation(Signal)and PCA figure were done for control quality of the gene chip data.The results showed good.2 Analysis results of chip data1)Number of significant differences genesAccording to the requirements of experiment,the genes showed significant differences according screened criteria of the | FC | must greater than 2,and Pvalue value less than 0.05.Results showed that compared with that of control group,the up-regulated gene number of LHS group was 1167,and the down-regulated gene number was 1337.The intuitived results were shown through volcano figure,scatter diagram and dendrogram.2)Pathway analyze of chip dataPathway enrichment analysis was made according to the screened different genes.The differences genes were enrichment analysis by all pathways in the genetic information in the KEGG and BIOCARTA.We selected ten pathways to show according to the P value.3)Signaling pathways histogram of chip dataSignaling pathways histogram showed the enriched condition of changed genes in the classic signaling pathways.Classical signaling pathways wereregulated by 800 different signaling pathways which collected by the IPA.All of the signaling pathways were used Log P to order.4)Signal path diagram of AMPKThe signal path diagram showed the effect of experimental data on signal transmission.The signal transmission process of various moleculars and up-down condition of different genes were given in the signal path diagram.The activation-inhibition projections for other genes were made according molecules activated prediction.The results showed transmission process of various moleculars of AMPK signal path.5)Upstream regulation analysis of changed genesThe upstream regulatory factors of all changed genes in the experiment were showed by upstream regulatory factors analysis table.Upstream regulatory factors can be any molecules which can affect the gene expression.They covered all types of molecules,including transcription factors,cytokines,small RNA,receptors,kinase,chemistry molecular and medicine.Activation z-score algorithm was used in IPA to predict the activation or inhibition of the upstream regulator,and decreased significantly prediction due to random data.The result showed hepatocyte nuclear factor-4?(HNF-4?)had been activated by prediction.6)HNF-4? upstream regulator network diagramUpstream regulator network diagram showed the relationships between upstream regulatory factors and downstream molecules which were related to the upstream regulator directly.3 Effects of LHS and CCl4-LHS on HNF-4? gene and protein expression levels of UCMSCs in vitroReal time-PCR results showed compared with those of control groups,the expression levels of HNF-4? increased significantly after LHS induced at different time points(P < 0.05).The expression levels increased with the extension of induction time,and expression level peaked in 7d group.Compared with those of control groups,the expression levels of HNF-4?enhanced significantly after CCl4-LHS induced at different time points.Among them,there were no significant differences of expression levels between 3d and 5d group.Compared with 3d and 5d group,expression level of 7d group increased significantly(P < 0.05).Compared with those of LHS groups of each time point,the expression leveles of HNF-4? increased significantly in CCl4-LHS groups(P < 0.05).Western blot results showed that compared with those of control groups,the expression levels of HNF-4? increased significantly at different time points of LHS groups(P < 0.05).The expression quantity increased with the extension of induction time,and the expression quantity was biggest in 7d group(P < 0.05).Compared with those of control groups,the expression levels of HNF-4? enhanced significantly at different time points of CCl4-LHS groups.Among them,the expression quantity peaked in 3 d,and then decreased(P < 0.05).Compared with those of LHS groups of each time point,the expression levels of HNF-4? increased significantly in CCl4-LHS groups(P < 0.05).4 Effect of BI6015 on differentiation of UCMSCs after CCl4-LHS induction in vitroReal time-PCR results showed compared with control group,the expression levels of HNF-4?,AFP,CK18,ALB and CYP3A4 enhanced significantly in CCl4-LHS-3d group(P < 0.05),and the expression levels of HNF-4?,AFP,CK18,ALB and CYP3A4 were lower significantly in BI6015group(P < 0.05),and the expression levels of HNF-4?,AFP,CYP3A4 were lower significantly in BI6015+CCl4-LHS group(P < 0.05),the expression levels of ALB enhanced significantly in BI6015+CCl4-LHS group(P < 0.05).Compared with CCl4-LHS-3d group,the expression levels of HNF-4?,AFP,CK18,ALB and CYP3A4 reduced significantly in BI6015 group and BI6015+CCl4-LHS group(P < 0.05).Compared with BI6015 group,the expression levels of AFP,CK18 and ALB enhanced significantly in BI6015+CCl4-LHS group(P < 0.05).5 Effects of CCl4-LHS on AMPK and LBK1 gene expression of UCMSCs in vitroReal time-PCR results showed that compared with control group,the expression levels of AMPK?1,AMPK?2 and LKB1 enhanced significantly in CCl4-LHS-3d group(P < 0.05).Western blot results showed that compared with control group,the expression levels of LKB1 and AMPK not changed significantly in CCl4-LHS-3d group,the expression levels of p-LKB1(S428),p-LKB1(T189)and p-AMPK were lower significantly(P < 0.05).6 Effects of AICAR and DM on differentiation of UCMSCs after CCl4-LHS induction in vitroWestern blot results showed that compared with those of control group,the expression levels of HNF-4? increased significantly in CCl4-LHS-3d group and DM group and CCl4-LHS+DM group,and were lower significantly in AICAR and CCl4-LHS+AICAR group(P < 0.05).Compared with those of CCl4-LHS-3d group,the expression levels of HNF-4? decreased significantly in AICAR and CCl4-LHS+AICAR group,and increased significantly in CCl4-LHS+DM group(P < 0.05).Compared with those of AICAR group,the expression levels of HNF-4? increased significantly in DM and CCl4-LHS+DM group(P < 0.05).Compared with those of DM group,the expression levels of HNF-4? reduced significantly in CCl4-LHS+AICAR group,and increased significantly in CCl4-LHS+DM group(P < 0.05).There were no significant differences of AMPK expression levels between groups.Compared with those of control group,p-AMPK expression levels reduced significantly in CCl4-LHS-3d,DM and CCl4-LHS+DM group,and increased significantly in AICAR and CCl4-LHS+AICAR group(P <0.05).Compared with those of CCl4-LHS-3d,AICAR group and CCl4-LHS+AICAR increased significantly,CCl4-LHS+DM group decreased significantly(P < 0.05).Compared with those of AICAR group,p-AMPK expression levels decreased significantly in DM and CCl4-LHS+DM group(P< 0.05).Compared with those of DM group,p-AMPK expression levels increased significantly in CCl4-LHS+AICAR group,and decreased significantly in CCl4-LHS+DM grou... |
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