| Part ⅠStudy on the regulation of cholestasis via MiR-125b-MRP2Objective: Through the animal model of cholestasis induced by ANIT,we explored the regulation mechanism of miR-125 b on the MRP2 pathway.Methods: Use ANIT to induce cholestasis modeling in mice and conduct animal experiments.The test groups were as follows:(1)using ANIT to induce cholestasis in mice,(2)normal mice injected with miR-125 b inhibitor to inhibit miR-125 b expression,(3)normal mice injected with miR-125 b inhibitor,and Use ANIT to induce cholestasis modeling in mice.(4)The control group is normal mice without any treatment.Observe the changes of serum liver function(ALT,AST,ALP,TB,DBIL)of the four groups of mice,and then perform HE staining on the liver tissues of the above mice to observe the liver pathology.At the same time,immunohistochemical staining,PCR test and Western Blot test were performed to check the expression of MRP2,miR-125 b,Ezrin,EBP50,F-actin,NF-κB,and STAT3.Results: Targetscan software analysis found that miR-125 b can target and regulate MRP2.An animal model of cholestasis induced by α-naphthyl isothiocyanate(ANIT).The changes of biochemical indexes and pathological manifestations of cholestatic liver injury caused by mice modeled at 75 mg/kg are similar to those of the IBC negative group after Kasai Children are most similar.The liver in the ANIT model group showed significant pathological damage,manifested as hepatocyte swelling,accompanied by inflammatory cell infiltration and obvious hepatocyte necrosis,and hepatocyte coagulation necrosis was seen in the tissue.The blood bilirubin and transaminase in the ANIT group increased sharply.Compared with mice in the control group and the ANIT model group,the expression of miR-125 b m RNA in the model group was significantly enhanced.In mice modeled by ANIT,the expression of MRP2 decreased significantly.There was no difference in the expression of NF-κB/STAT3 in the four groups of mice,but the expression of phosphorylated NF-κB/STAT3 was significantly increased in the cholestatic mouse group,and the expression of Ezrin,EBP50 and F-actin in the cholestatic mouse group was also Significantly enhanced.Conclusion: An animal model of cholestasis induced by α-naphthyl isothiocyanate(ANIT).The liver inflammation is activated to regulate the expression of miR-125 b.On the one hand,it directly targets the m RNA of MRP2 to cause changes in its protein expression.On the other hand,it regulates the localization of MRP2 to the cell membrane by acting on F-actin,which causes and aggravates it.Hepatic cholestatic reaction.Part ⅡStudy on the regulation of IBC(+)and IBC(-)via MiR-125b-MRP2 in pediatric patientsObjective: To analyze the expression of MiR-125b-MRP2 in IBC(+)and IBC(-).Methods: A study was conducted with 50 BA patients who had experienced KP and liver transplantation(LT),the clinical and imaging data were collected from January 2008 to December 2018.As confirmed by CT examination,we divided those patients into IBC(+)group who had bile duct expansion,and IBC(-)group who did not had bile duct expansion.Results: 50 patients’ were selected for HE staining(including 18 in IBC(+)group and 32 in IBC(-)group),the degree of bile plug in the IBC(+)group was lower than that in the IBC(-)group(P value = 0.013).We compared found that there were irregular bile lakes with bile-like crystals surrounded by fibrous tissue,and severe fibrosis in the IBC(+)group,no typical bile duct epithelioid structure was observed.But the degree of liver fibrosis and bile duct hyperplasia and the incidence of bile duct plate malformation between two groups had no significant difference.The presence of IBC after KP is not significantly related to the occurrence of cholangitis(P value = 0.878).We detected MRP2 protein expression in tissues from IBC(+)and IBC(-)with immunohistochemistry.We found MRP2 protein was highly expressed in IBC(+)group.However,the m RNA level of MRP2 had no difference in these two groups.Further staining of 50 liver pathological tissues revealed that the expression of miR-125 b in the IBC(+)group was significantly reduced.Conclusion: MRP2 was highly expressed in IBC(+)tissues,may play an important role in the formation and treatment of IBC.Part Ⅲ Clinical study on the relationship between IBC and cholangitis,native liver survival time and liver functionObjective:To analyze the pathological changes and the formation of intrahepatic biliary cysts(IBC)in children with biliary atresia(BA)during the period of post Kasai procedure(KP)to native liver failure,and to explore its correlation with cholestasis,and explore the relationship between this pathological change and native liver survival(NLS)time.Methods: A study was conducted with 179 BA patients who had experienced KP and liver transplantation(LT),the clinical and imaging data were collected from January 2008 to December 2018.As confirmed by CT examination,we divided those patients into IBC(+)group who had bile duct expansion,and IBC(-)group who did not had bile duct expansion.Results: There were 27 female patients(27/36,75%)in the IBC(+)group,which was higher than the 77 patients(77/143,53.5%)in the IBC(-)group,the difference was statistically significant.As for the comparison of the average KP age and the incidence of postoperative cholangitis in two groups,there were no significant difference(both P value > 0.05).The median NLS time of IBC(+)group(11.50 months)was longer than that of IBC(-)group(9.00 months).NLS time in the IBC(+)group was better than that in the IBC(-)group(P value=0.038).Liver function index comparison was found that the IBC(+)group were all lower than those in the IBC(-)group except TBA and LDH,and the levels of total bilirubin(TB)and direct bilirubin(DB)were significantly different between the two groups(P value < 0.05).Conclusion: IBC after Kasai operation for biliary atresia may be a compensatory manifestation,especially in some children with IBC who have cysts communicating with bile and intestine.Cholagogic and anti-inflammatory therapy may temporarily reduce bilirubin level and improve jaundice. |
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