| Objective: To explore the contribution of regulation T cell (Treg), Th17 and dendritic cells (DC) in progressive necroinflammatory of biliary atresia. To develop an animal model of biliary atresia(BA) , with the Rhesus Rotavirus (RRV),to investigate the effection of hindrance of Th17 cells differentiation to attack rate of biliary atresia (BA).Methods: 50 patients were divided into 2 groups: biliary atresia (BA, n=32), control group with no diseases of liver (C, n=18). The expression of Foxp3, IL-17, R0R-γt mRNA and protein in livers were detected with Q-RT-PCR and immunohistochemistry; The frequencies of CD4+CD25+Foxp3+T and Th17 cells in peripheral blood were detected by flow cytometric analysis; The expression of IL-10, TGF-β, IL-17, IL-6, IFN-γ, IL-12 in blood serum were detected with ELISA; Culture DC,CD4~+T and CD8~+T cells from peripheral blood, to detected the expression of IL-6 in medium with ELISA. Mixed culture T cells and DCs, to detected the expression of IL-17 in medium with ELISA. The expression of TLR3, TLR7, TLR8 protein in DC were detected with Q-RT-PCR, Western blot and immunofluorescence; New born Balb/c mice were randomly devided into 3 groups. Group R as mice were infected with RRV intraperitoneolly, group N as mice were given 2% Fetal Cow Serum-Minimum Essential Medium (2%FCS-MEM) as control. Group B as mice were infected with RRV intraperitoneolly, and followed by being infected with IL-6 neutralization antibody in 24h after RRV infected. In Group B, there were 4 group devided according to the dose of IL-6 antibody, they are 5μg, 15μg, 20μg, 25μg. The clinical and histomorphologic changes associated with BA were observed and scored, the extrahepatic bile ducts were serially sectioned and stained with Hematoxylin-Eosin stain (HE). Observe the mRNA expression of Foxp3, IL-17, R0R-γt in the liver of animal model of BA with the help of Real-Time PCR(Q-RT-PCR) . Culture DC,CD4+T and CD8+T cell from spleen, to detected the expression of IL-6 in medium with ELLISA. mixed culture T cells and DCs, to detected the expression of IL-17 in medium with ELLISA. The expression of TLR3, TLR7, TLR8 protein in DC were detected with Q-RT-PCR.Results: The expression of Foxp3,IL-17,R0R-γt in liver is significantly higher in BA than in C (P<0.05); The frequencies of Th17 is significantly lower in BA than in C (P<0.05),but the frequencies of CD4+CD25+Foxp3+T is not different between BA and C group (P>0.05); however the radio of Treg/Th17 is significant lower in BA than in C(P<0.05). The expression of IL-10,TGF-β, IL-17,IFN-γ,IL-12,IL-6 in blood serum is significantly higher in BA than in C (P<0.05); The expression of IL-6 in medium of DC is significantly higher in BA than in C (P<0.05); The expression of IL-17 in medium of PMBC + DCs + TGF-βof BA is higher (P<0.05); The expression of TLR3, TLR7, TLR8 protein in DC is significantly higher in BA than in C (P<0.05); Of the 94 mice from group R, 64 (71.91% ) mice showed signs of cholestasis and growth retardation. Hepatobiliary anatomy found atrophy or dilation of gall bladder and extrahepatic biliary atresia, which resembles the BA of human being, 6.25% of mice's life time was over 21d, no mice recovered. 193 mice from group B, were devided into 4 groups according to the dose of IL-6. There were 48 mice in 5μg, 15μg, 20μg group respectively. And there are 34(73.91%), 33(70.21%), 29(61.71%)mice showed signs of the above. There were 49 mice in 25μg group and only 25(52.08%)showed signs of the above, significant lower than group R (P<0.05). 9.52% of mice's life time was over 21d. 16% of mice recovered, significant higher than group R (P<0.01). None of the31 mice from group N showed signs of the above after inoculation with MEM.The expression of IL-17,ROR-γt mRNA is significant lower in livers from 25μg group and group N than in group R (P<0.05). The expression of IL-6 in medium of DC is significantly higher in group R than in group N (P<0.05); The expression of IL-17 in medium of T cells + DCs + TGF-βof BA is higher (P<0.05); The expression of TLR3, TLR7, TLR8 mRNA in DC is significantly higher in group R than in group N (P<0.05);Conclusion:1. the expression of Treg, Th17 cells and TLRs in biliary atresia is significantly higher in BA, but the ratio of Treg/Th17 from blood decreased, which indicates that Treg, Th17 cells and TLRs play important roles in BA.2. Maybe the activation of DCs from BA by TLRs pathway, secreting IL-6, which promotes the differentiation of Th17, that is play an important role in BA.3. We reduced the differentiation of Th17 cells by infecting IL-6 antibody. The morbidity of animal models is significant lower. The findings indicate that Th17 plays a key role in BA. This presents a new basis for further studies in the pathogenesis of BA .
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