| Biliary atresia(BA)is a common disease causing obstructive jaundice in children.It has a poor prognosis and a high mortality.Its typical pathological feature is progressive inflammatory response centered on bile duct.However,the etiology of BA is still unclear.Studies have shown that some genetic factors are closely related to the occurrence of BA.Our previous GWAS study showed that there was a significant difference in STAT3 genotype between BA children and normal controls.So,how does STAT3 affect BA?What is its mechanism?Objective:1.To verify the gene loci with significant differences that found by the genome-wide association study of BA children and make its functional predictions,and to detect the expression of STAT3 in the liver of BA children;2.To study the effect of STAT3 expression on BA and its mechanism,so as to provide a new way to explore the pathogenesis of biliary atresia.Methods:This topic is divided into two parts:Part one:Verifying the genetic susceptibility of STAT3 and detecting its expression in the liver of children with BA.1.Using gene mass spectrometry to verify the abnormal genes of BA found in the previous study and to predict its function;2.Real-time PCR,Western Blot and immunohistochemical methods were used to detect and compare the expression of STAT3 in the liver of choledochal cyst and BA children from mRNA and protein levels.Part two:The effect of down-regulation of STAT3 expression on BA and its mechanism1.BA mice were induced by rotavirus(RRV)and injected with STAT3 inhibitor(Stattic)/activator(Colivelin)intraperitoneally.The mice were divided into four groups(normal control group,RRV group,Stattic + RRV group,Colivelin + RRV group)and observed at different growth points(postnatal 3,6,9,12days),The survival rate,body weight,skin jaundice and the development of extrahepatic bile duct were observed.HE staining was used to observe the infiltration of inflammatory cells in the liver portal tracts of each group.2.Flow cytometry was used to detect inflammatory cells(NK cells,CD4+T cells,CD8+T cells,macrophages,neutrophils)in the liver portal tracts at different growth points(3d,6d,9d after birth)in BA mice.The content of neutrophil(PMN)in the liver portal tracts of different groups(3d,6d,9d after birth)was detected by immunohistochemistry.3.Immunohistochemical method was used to detect the PMN content in the liver portal tracts of the normal control(portal cavernous degeneration)group,disease control(choledochal cyst)group and BA children.4.Human bile duct endothelial cells and PMN were co-cultured in vitro and divided into three groups(normal control group,BEC+PMN group,BEC+PMN+PMN activator group)to observe the effect of PMN on BEC;flow cytometry was used to detect the apoptosis of BEC at different time points(24h,48h)in each group.5.Chemokines(IL-8,IL-2,IL-4,IL-6,CXCL-2,CXCL-13,CCL-3,CCL-11)in peripheral blood of three groups(normal control group,cholestasis group,BA group)were detected according to the instructions of Bio-plex Pro Human Chemokine Assays.6.In vitro,the expression of IL-8 in human BEC was detected by qPCR and ELISA after addition of STAT3 inhibitor.Results:The results are divided into two parts,Part one:There were polymorphic changes in IL-10,IL-18 and STAT3 genes in BA patients,and their susceptibility loci were known to express quantitative trait loci,which could regulate the expression level of corresponding genes.Compared with children with choledochal cyst(CC),the mRNA and protein levels of STAT3 gene decreased significantly(p<0.01).Part two:1.To further investigate the effect of downregulation of STAT3 on BA,we injected Stattic and Colivelin into the peritoneal cavity of BA mice.The results showed that compared with RRV group,jaundice in Stattic+RRV group gradually increased and the average body weight decreased significantly(p<0.05),the survival rate is reduced,the extrahepatic biliary atresia time is advanced,and the inflammatory cell infiltration in the liver portal tracts is significantly increased.It is suggested that the down-regulation of STAT3 expression may aggravate the inflammation of hepatic portal tracts and affect the growth and development of BA mice.2.To further understand the types and expression of inflammatory cells that play an important role in the liver portal tracts,we used flow cytometry and immunohistochemistry to detect and found NK cells,CD4 T cells,CD8+ T cells,macrophages and PMN were all expressed in the acute phase of BA,but mainly neutrophils.Compared with RRV group,the number of PMN infiltration in the hepatic portal tracts of Stattic+RRV group increased significantly(p<0.05).It was suggested that the downregulation of STAT3 expression could significantly increase the number of PMN infiltration in the hepatic portal tracts of BA mice.3.In order to clarify the degree of PMN infiltration in the liver portal tracts of children with BA,we used immunohistochemistry method to detect and found the results:compared with the normal control(portal cavernous degeneration)group,disease control(CC)group,the number of PMN in the hepatic portal tracts of BA children increased significantly(p<0.01).It is suggested that PMN may play an important role in the pathogenesis of BA.4.In order to clarify the effect of PMN on human BEC,we used in vitro cell co-culture and found that activated PMN can directly damage BEC.The results of flow cytometry showed that there was no significant difference in BEC apoptosis among the three groups after co-culture for 24h.Compared with BEC group and BEC+PMN group,the number of BEC apoptosis in BEC+PMN+PMA group was significantly increased after co-culture for 48h(p<0.01).It suggests that activated PMN can cause apoptosis in human BEC and cause bile duct injury.5.In the acute stage of inflammation,chemotaxis is most important for the number and function of PMN.Therefore,we measured the neutrophil chemokines,the results showed that:compared with the normal control group(cutaneous hemangioma)group,the peripheral blood serum IL-8 levels in BA patients were significantly increased(p<0.001);compared with the disease control(cholestasis)group,the peripheral blood serum IL-8 levels in BA patients were also significantly increased(p<0.05).However,the levels of IL-2,IL-4,IL-6 and other chemokines were not significantly different in the peripheral blood serum of the three groups.It is suggested that PMN infiltration increased in the hepatic portal tracts of children with BA may be caused by the increased expression of IL-8.6.In order to clarify whether the down-regulation of STAT3 may affect the secretion of IL-8 by human BEC,we co-cultured human BEC+Stattic in vitro and detected it by qPCR.The results showed that the content of IL-8 produced by BEC increased significantly when Stattic was added and IL-8 content increased with the increase of Stattic concentration.ELISA confirmed that Stattic could promote the production of IL-8 in BEC,and IL-8 content increased with the increase of Stattic concentration in the early stage of reaction(<6h).This indicates that down-regulation of STAT3 expression may lead to increased IL-8 secretion by human BEC.Conclusion:STAT3,a genetic susceptibility gene for biliary atresia,promotes the aggregation of neutrophils in hepatic portal tract by up-regulating the expression of IL-8,thus aggravating the apoptosis of BEC,leading to bile duct injury and participating in the occurrence of BA. |
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