The Sequencial Changes Of Hepatic Fibrosis Relevant Genes In Rat Biliary Atresia Induced By Bile Duct Ligation

ObjectiveBiliary atresia(BA)is a severe neonatal hepato-disease involving external and intrahepatic bile duct in infancy which results in liver cirrhosis in the end.The feature of BA remains progressive inflammation,obstruction,fibrosis of the biliary system, then disappeared in the end.Hepatic fibrosis procession will still to be progress, even though reconstruction operation was administrated.About 80%of patients will need liver transplantation eventually.There have been studies which prove that epithelium-mesenchymal transition(EMT)play an important role in the fibrosis processes of various organizations,including renal fibrosis,lung fibrosis and so on.It was found that EMT was accompanied by the reactivation of some embrycgenesis related gene.Therefore,in this study we will ligate rat bile ducts to mimick neonatal biliary atresia in human being;to investigate the correlated molecular mechanism of MET in liver fibrosis.Materials and Methods1,Animal Models100 healthy wistar rats,about three weeks old,weight ranged from 30 to 40g, obviously abnormalities were excluded.They were randomly divided into two groups, BDL group(50) and control group(50).External bile ducts of BDL group were ligated and mutilated near hepatic portal,while control group received sham operation.The two groups were bred under identical conditions after operations.2,Experiment materialsThe animals were sacrificed at 3d,7d,14d,21d and 28d postoperatively,complete livers were taken out.Identical lobes were taken out as experiment samples,parts were fixed in 4%paraformaldehyde for immunohistochemisty,the other part is frozen in -80℃for RT-PCR.3,RT-PCRTo detect the mRNA levels of inflammation relevant gene TGF-β1,early fibrosis relevant gene COL1,epithelial specific cytokeratin CK19 and fibrosis cellular molecularα-SMA with RT-PCR.ResultGross appearance of specimenThe urine of BDL group appeared yellow after two days of operation.At 3 days after ligation,small lobes near the portal exhibited hydropic degeneration.At 7 days, these small lobes became yellow adipose degeneration besides further hydropic degeneration,at 14 days,the hydropic degeneration disappeared,the adipose degeneration extended to larger lobes.At 21 days,many foxtail millets could be seen at the surface of the livers,adipose degeneration became disappear.At 28 days,foxtail millets became more obvious,the volume of the livers decreased and the appearance changed into yellow.In control group,the hepatic lobes was ruddy and the surface was smooth and glossy all the time,the volume increased gradually with the growth of the animals.Result of RT-PCRRT-PCR amplification of relevant genes showed positive bands in both sham operation group and BDL group.The RT-PCR amplifications of relevant genes were weaker in the control group,but generally bright in BDL group.In BDL group,the mRNA levels of genes increased gradually with time,especially the mRNA levels of TGF-β1 and COL1.There was no marked change in control group.According the statistical analysis,the amount of TGF-β1 and COL1 mRNA became to have significant differences from the 3rd day of operation.There's significant difference of CK19 between the 2 groups at 21 days and 28 days.There's significant difference ofα-SMA between the 2 groups from 7 day after operations.In BDL group,the expression of CK19 gene have no significant difference from 3days to 7days anct from 7 days to 14days after BDL,the expression ofα-SMA have no significant difference from 3days to 7days after BDL,except these,the expression of other genes were all different at different days after BDL.All the genes expressions have tendency of increase at different day groups,which was obvious from 14 day to 21day.especiallyα-SMA.ConclusionAfter BDL,livers of animals appeared progressed fibrosis.We found the expression of fibrosis relevant genes were higher in the BDL group than that of control group.Furthermore,the expression increased with the days of ligation.Moreover,we discovered that the period of 14d to 21d was a crucial duration for the development of hepatic fibrosis,which may be an important time for the epithelium of bile duct began to exhibit fibroblast specific characteristics through the procession of EMT.The increase of COL1 may appear earlier thanα-SMA at hepatic fibrosis,so COL1 could be used as an early indication for liver fibrosis.In the development of liver fibrosis, inflammation relevant factor TGF-βmay play important roles.