Clinical Features Of Autoimmune Hepatitis Patients With Poor Response To Treatment And In Vitro Study Of UC-MSCs Therpy

Objective:To investigate the clinical features of autoimmune hepatitis?AIH?patients with poor response to treatment.To investigate the mechanism of umbilical cord-derived mesenchymal stem cells?UC-MSCs?in the regulation of regulatory T cells?Treg?and T helper 17 cells?Th17?in patients with autoimmune hepatitis by coculturing UC-MSCs with peripheral blood mononuclear cells?PBMC?from AIH.Method:1.A total of 61 AIH patients were enrolled,among whom 49?80.33%?achieved complete response and 12?19.67%?had incomplete response.The two groups were compared in terms of clinical manifestations,laboratory makers,abdominal ultrasound findings,pathological features by liver biopsy.Binary logistic regression analysis was used to determine influencing factors.2.The PBMC of AIH and healthy control were collected to compare the proportions of Treg and Th17 in the two groups.Then,PBMC from AIH patients cocultured with UC-MSCs for 72h at ratios of 1:1,5:1,10:1,respectively.a.The proportions of Treg and Th17 cells were detected by flow cytometry;b.The relative mRNA expression of FOXP3 and RORC were assessed by qRT-PCR;c.The cytokines in the supernatants were measured by ELISA;d.Inhibitors targeting TGF-?1 and PGE2were added to the coculture system,and the percentages of Treg and Th17 cells and the relative mRNA expression of FOXP3 and RORC were observed.Results:1.The clinical features of refractory autoimmune hepatitis1.1 The patients with incomplete response were characterized by significantly higher serum levels of ALT,AST,ALP,GGT,TBIL,IgG and IgM at diagnosis.1.2 Patients with incomplete response were distinguished for a higher positive rate of autoantibodies except ANA,SMA,AMA/M2?75%vs16.3%,P<0.001?,and there was a significant difference in the positive rate of gp210?25%vs 0%,P<0.01?.1.3 The proportion of cirrhosis?50%vs 16.3%,p<0.05?and splenomegaly?58.3%vs 22.4%,p<0.05?were higher at diagnosis in patients with incomplete response.1.4 The binary logistic regression analysis showed that high serum level of ALP?P=0.034,OR 1.017,95%CI 1.001-1.033?,seropositivity for autoantibodies except ANA,SMA,AMA/M2?P=0.017,OR 70.842,95%CI 2.132-2354.371?,and cirrhosis?P=0.049,OR 28.777,95%CI 1.015-815.854?were independent risk factors for initial treatment outcome.2.The mechanism of UC-MSCs in regulation of peripheral Treg and Th17 in AIH2.1 Compared to the healthy control group,the level of Treg cells in AIH patients were significantly decreased?3.008±0.2753 vs 1.693±0.2493,P<0.05?,and the level of Th17 cells were significantly increased?1.983±0.2158 vs 3.708±0.1930,P<0.05?.2.2 UC-MSCs up regulated Treg proportion?1.693±0.2493 vs 4.480±0.4899,P<0.05?and reduced the proportion of Th17 cells?3.708±0.1930 vs 1.725±0.1216,P<0.05?dose-dependently,which was not depended on cell-cell contact.2.2 UC-MSCs up regulated the relative mRNA expression of FOXP3?1.005±0.001528 vs 3.276±0.3868,P<0.05?and down regulated the relative mRNA expression of RORC?1.002±0.001222 vs 0.4247±0.05456,P<0.05?.2.3 Supernatant TGF-?1,IL-6 and PGE₂ level increased,IL-10 and IL-17A decreased after coculturing,and the difference was statistically significant?P<0.05?.2.4 TGF-?1 antibody mitigated UC-MSC mediated up-regulation of the proportion of Treg cells and the gene Foxp3 expression,and PGE₂ inhibitors mitigated UC-MSC mediated down-regulation of the proportion of Th17 cells and RORC gene expression,and the difference was statistically significant?P<0.05?.Conclusion:1.High serum level of ALP,positive autoantibodies except ANA,SMA,AMA/M2 and liver cirrhosis are closely associated with poor response in AIH patients.2.UC-MSCs up regulated Treg proportion and reduced the proportion of Th17 cells in AIH patients dose-dependently,which was not depended on cell-cell contact.3.UC-MSCs up-regulate Treg through TGF-?1 and down-regulate Th17 cells through PGE₂ in patients with AIH.