| PART I EFFECT OF HIGH CONCENTRATION OF BILE ACID OR SALT ON THE PROLIFERATION AND APOPTOSIS OF HEPATIC STEM CELLS HP14-19Objective: To research the effect of high concentration of toxic bile acid or salt on the proliferation and apoptosis of hepatic stem cells HP14-19 cells and the regulation mechanism of apoptosis-related signaling pathways.for transplanted hepatic stem cells HP14-19 being easier Survival into the cholestatic microenvironment providing theoretical and experimental evidence in vivo.Methods:1.Embryonic hepatic stem cell HP14-19 cells were used as the research object,CCK-8 kit was used to detect bile salts’ Effect on the viability of HP14-19 cells at different concentrations(0,100,200,400,800,1200 μmol/L)and at different times(6 h,12 h,24 h,48 h).2.The effect of bile salts on the proliferation of HP14-19 cells at different concentrations(0,100,200,400,800,1200 μ mol/L)and at different time points(6h,12 h,24h,48h)was detected by crystal violet staining.3.The apoptosis of HP14-19 cells treated with bile salts was detected by flow cytometry and DAPI staining.4.Western-blot were used to detect the expression of related markers of apoptosis signal pathway in bile salts after treatment of HP14-19 cells at the nucleic acid level and protein level.Results:1.CCK-8 cell viability assay showed after treated with different concentrations of bile salts HP14-19 cells for 1 day,when the bile salt concentration is about 500μmol/L,cell survival rate is close to about70%;and when HP14-19 cells treated with 500 μ Mol/L GCDC for6 h,12h,24 h and 48 h,the cell survival rate was found to be close to about60%.so,the research of protocol was suitable.2.Crystal violet staining showed that after treated with different concentrations of bile salts on HP14-19 cells for 1 day,the number of cells decreased with the increase of bile salt concentration.3.The results of flow cytometry showed that the apoptosis rate of HP14-19 cells was significantly higher after treatment with GCDC compared with the control(P<0.05);DAPI staining showed Nuclear condensation and Nuclear fragmentation after GCDC treatment of HP14-19 cells.4.Western-blot detection found that treatment group was significantly higher than the control group in the apoptosis signal pathway related markers Caspase-3 and Bax expression while Bcl-2 expression was the opposite(P <0.05).Conclusion:1.High concentration of bile acid or salt can inhibit the proliferation of hepatic stem cells HP14-19 with concentration and time dependent,and regulate the apoptosis of hepatic stem cells by down-regulating Bcl-2 gene and up-regulating Caspase-3 and Bax genes.PART II STUDY ON THE EFFECT OF MICROENVIRONMENT FACTORS DERIVED FROM CHOLESTATIC CIRRHOSIS ON HEPATIC STEM CELL DIFFERENTIATIONObjective:This study investigated the expression of cytokines from the pathological microenvironment of cholestasis liver cirrhosis at different time points in bile duct ligation(BDL)mice.In vitro,we are striving to find the optimal combination of cytokines to efficiently induce hepatic stem cells HP14-19 to differentiate into mature hepatocytes.Methods:1.100 children with biliary atresia at random were selected as experimental group and 20 children with choledochal cyst as control group.Serum AFP(alpha-fetoprotein)expression was detected in these cases;The liver tissues of 20 children with biliary atresia and 20 children with common bile duct cysts were examined by immunohistochemistry for the expression of stem cell markers ov-6 and c-kit.2.The pathological microenvironment of cholestatic cirrhosis was simulated by 8-week-old male Balb/c mouse with Bile Duct Ligation(BDL)operation.3.Mice Liver tissues of 3d,7d,14 d,and 21 d in the model group and sham group were selected,fixed,dehydrated,embedded and sliced in 4%paraformaldehyde;Immunohistochemical staining was used to detect the cells in the mouse liver tissue.The expression of EGF(epidermal growth factor),HGF(hepatocyte growth factor)and TGF-α(alpha-transforming growth factor).4.The HP14-19 cells of mouse embryonic hepatic stem cells were used as the research object.The luciferase reporter gene was used to detect ALB-Gluc activity at different time points.5.qRT-PCR and Western-blot were used to detect the expression of hepatocyte-related markers AFP(alpha-fetoprotein),CK18(Cytokeratin-18)and ALB(Albumin);indocyanine green(ICG)and periodic acid-Schiff(periodic acid-schiff,PAS)staining detects the mature function of liver cells.Results:1.Compared with the level of serum AFP in children with common bile duct,the serum AFP value in children with biliary atresia was significantly higher(P<0.05);the stem cell markers OV-6 and C-kit were significantly higher in the liver than in children with biliary atresia.Children with common bile duct cysts were high(P < 0.05).2.Total bilirubin,DBL(direct bilirubin),ALT(Alanine aminotransferase),and AST(Aspartic aminotransferase)were significantly increased compared with the sham-operated group after ligation of common bile ducts in mice(P<0.05);Masson staining showed that Fibrosis was aggravated with prolongation of ligation time;Western-blot was used to detect fibrosis-specific α-SMA expression with prolonged ligation time(P<0.05).3.Compared with the control,EGF expressed more around hepatocytes and bile ducts,and peaked at 7-14 days;HGF,TGF-α and EGF were similar and peaked at 14 days;14 days later,the expression of cytokines may be reduced which Associated with cell necrosis in later stages.4.After different concentration(0,5,10,20,40,80 ng/ml)cytokines treated HP14-19 cells alone for 2d,4d,and 6d,It showed that The induction effect was better when EGF is at 10ng/ml,HGF at 20ng/ml and TGF-α at 20ng/ml(P<0.05);when induced by EGF/HGF,EGF/TGF-α,HGF/TGF-α,EGF/HGF/TGF-α of three kind of cytokines after 3 days,6days,and 9 days,It showed that the induction effect was best at the combination of 10 ng/ml EGF,20 ng/ml HGF and 20 ng/ml TGF-α(P<0.05).5.When the cytokines were treated with HP14-19 cells for different time according to the above combination,the results of q RT-PCR and Western-blot showed that the expression of hepatocyte-associated markers AFP,CK18,and ALB increased as the induction time prolonged;the results of PAS and ICG staining showed that the cell maturation function is enhanced.(P<0.05)Conclusion:1.Ligation of the common bile duct of mice can effectively simulate cholestatic cirrhosis,which provides a good animal model for our research.2.The protective cytokines derived from the pathological microenvironment of cholestatic cirrhosis have a good hepatic differentiation effect on hepatic stem cells and can stably maintain hepatocyte function.3.In the combination of EGF 10 ng/m L,HGF 20 ng/m L,and TGF-α20 ng/m L,the effect of promoting hepatic differentiation was higher,which has certain guidance for the further study of cytokines combined with liver stem cell transplantation for the treatment of cholestatic cirrhosis.significance. |
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