| Objective:To explore the abnormal changes of T lymphocyte subgroups、B lymphocyte subgroups and regulation T cells in peripheral blood and their correlations with liver dysfuncion, immunoglobulin and hepatitis activity index(HAI) in patients with autoimmune hepatitis (AIH), understanding the mechanism lymphocyte subsets acting in the pathogenesis of AIH. To test the ratio of the expression of CD3positive cells、Interferon-γ (IFN-γ) positive cells、Programmed death-1(PD-1) positive cells and Programmed death ligand-1(PD-L1) positive cells in hepatic tissue. Statistically analyze their changes before and after MSCs transplation in the treatment of AIH mice model, discussing the role of PD-1pathway played on MSCs treatment of AIH.Methods:1Twenty patients were selected as group AIH, who were diagnosed as AIH and not accepting corticosteroid treatment. Twenty healthy subjects were brought into control group. The two groups all received tests of liver function, consisting of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), y-glutamyltransferase(y-GT), Total bilirubin(TBIL), Direct bilirubin(DBIL), and immune function, including immunoglobulin G(IgG), immunoglobulin M(IgM) immunoglobulin A(IgA). Moreover, liver biopsy and pathological examination were carried out to evaluate hepatitis activity indices (HAI) for AIH group. The subsets of T、B lymphocytes and regulatory T cells(Tregs) were detected by flow cytometry(FCM). Correlations between lymphocyte subgroups and indicators of liver function, immunoglobulin and HAI were evaluated.2Liver tissue specimens from AIH model mice and normal mice were divided into three groups, group A were treated by MSCs, group B were treated with PBS, and group C was control group. This research has three time points:modeling,1week after modeling,4weeks after modeling. At each point, mice liver tissue specimens in A, B, and C groups were test the positive expression of CD3, IFN-y, PD-1, PD-L1using immunohistochemical method. The percentages of positive cells were recorded, and the results were statistically analyzed.Results 1The ratio of T, B lymphocyte subgroups and Tregs in peripheral blood and their correlations with liver dysfuncion, immunoglobulin and HAI of AIH patients and control group.1.1The average percentage of CD3+CD4+T, CD3+CD8+T, CD19+B, CD5+CD19+B cells, and the ratio of CD5+CD19+B/CD19+B in peripheral blood of AIH patients were all significantly higher in AIH group than in the control group(P<0.05), but there were no statistical significance between group AIH and control group in The average percentage of CD3+T, CD4+CD25+CD127dim cells, and the ratio of CD3+CD4+T/CD3+CD8+T (P>0.05).1.2The average lever of ALT, AST, ALP, y-GT, TBIL DBIL, IgG of these two groups have significantly statistically differences (P＜0.01); The values of IgM, IgA in AIH group have no statistically difference from normal group(P>0.05). The HAI scores7.60±4.51in AIH group.1.3The percentages of CD3+CD4+T, CD3+CD8+T cells had a positive correlation with ALT, AST and HAI (P<0.01), but no relevance with the ALP, GGT, TBIL and DBIL (P>0.05). In addition, the ratio of CD5+CD19+B cells was positively related to ALT, AST, HAI and IgG (P<0.05), but had no significant relevance with IgM and IgA (P>0.05).2The percentage of positive expression of CD3+, IFN-γ, PD-1, PD-L1in each group’s mice liver tissue specimens at different time points.2.1In the time of modeling,1week after modeling, and4weeks after modeling, The percent of CD3positive cells in group A were36.00±5.07%,27.94±2.33%and21.58±4.60%, compared in this group, the difference was statistically significant (P <0.05), compared with the time of modeling, the values of the1week and4weeks respectively decreased by22%and40%. In the1and4weeks, there was a statistically significant difference between group A and group B (P＜0.05).2.2In the3time points, the percentage of IFN-y positive cells in group A were30.06±5.19%,26.52±3.88%,20.86±3.35%, compared with the modeling week, the values of the1and4weeks respectively decrease12%and31%, and the differences was statistically significant (P＜0.05). In the1and4weeks, there was a statistically significant difference between group A and group B (P＜0.05).2.3In the3time points, the percentage of PD-1positive cells in group A were17.92±4.29%,13.24±2.71%and10.98±2.47%, compared with the modeling week, the values of the1and4weeks espectively decreased by26%and39%, showing a statistically significant difference(P<0.05). In the1and4weeks, there was a statistically significant difference between group A and group B (P<0.05).2.4In the3time points, the percentage of PD-L1positive cells in group A were3.12±1.19%,8.94±3.39%and12.38±4.20%, compared to the group, the differences were statistically significant (P＜0.05). Compared with the modeling week, the percentage of PD-L1positive cells in the1and4weeks respectively increase1.9times and3times. In the1and4weeks, there was a statistically significant difference between group A and group B (P<0.05).Conclusion1The percentage of CD4+T cells, CD8+T cells, CD19+B cells, CD5+CD19+B cell and CD5+CD19+B/CD19+B ratio in Peripheral blood of AIH patients were significantly higher than the controls, and had a positive correlations with the lever of ALT, AST, IgG and HAI score, suggesting that the abnormalities of T, B lymphocytes were consistent with the degree of liver damage.2The percentage of Tregs of AIH patients was equivalent to the control, indicating that the number of Tregs did not paly an important role in the pathogenesis of AIH.3After the treatment of MSCs transplantation, liver CD3+, IFN-γ and PD-1positive cells of AIH were significantly decreased, while the expression of PD-L1positive cells was significantly increased, indicating that MSCs cure AIH may be through inhibiting the expression of cytokines and regulating PD-1pathway. |
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