| Objective To investigate of expressions and function of the Th9 (T-helper 9) cells in patients with hepatitis B liver cirrhosis, to investigate of phenotype of the Th9 cells in patients with hepatitis B liver cirrhosis as well as Macrophage inflammatory protein 3 a (MIP-3a, CCL20)/CC chemokine receptor 6 (CCR6) axis of Th9 cells may influence. To explore whether they are involved in the pathogenesis of liver fibrosis.Methods Peripheral blood and liver tissue from 30 cases of patients with hepatitis B cirrhosis and 10 cases of healthy donors were recruited. Liver biopsies or surgical removal of the specimen were collected from 18 HBV-LC patients and from 6 patients with healthy livers. The proportion and phenotype of IL-9+IL-22-CD4+T(Th9) cells in the peripheral blood was tested by Flow cytometry. Serum Interleukin-9 (IL-9) and CCL20 levels were tested by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining was used to detect a-smooth muscle actin (a-SMA), CCR6, CCL20 protein in liver tissues paraffin section respectively. Then the correlation analysis between the percentage of Th9 cell、IL-9 as well as CCL20/CCR6 chemotactic axis and liver fibrosis were performed.Results (1) Flow cytometry test showed that the proportion of peripheral blood IL-9+IL-22-CD4+T(Th9) cells (3.55%±1.69%) in hepatitis B cirrhosis group was significantly increased as compared to normal control group (0.97%±0.24%). The percentages of CCR4+Th9 cells、CCR6+Th9 cells in Th9 cells in hepatitis B cirrhosis group were significantly increased as compared to normal control group (respectively:85.8%±12.5% vs.56.4%±2.36%; 78.1%± 10.6%vs.56.4%±9.04%), the difference were statistically significant (all P<0.05). (2) The enzyme-linked immunosorbent assay test results shows that: Compared with normal control group, significant elevation of serum IL-9 level and CCL20 level were demonstrated in the hepatitis B cirrhosis group, (respectively:25.55±4.62 vs.16.18±1.32 pg/ml; 224.77±33.73 vs.34.58±3.72 pg/ml), the difference were statistical significant (all P<0.05). (3) Immunohisto-chemical staining analysis results showed that the expression of CCR6 in the membrane of infiltrated lymphocytes in the portal area, the expression of CCL20 in the cell membrane of dendritic cells, hepatic stellate cells, fibroblasts, endothelial cells and cytoplasm of epithelial cells of bile duct in the portal area. Compared with normal controls, the expression of CCR6 and CCL20 in hepatitis B cirrhosis group were significantly increased (respectively:0.027±0.021 vs. 0.006 ±0.001,0.033±0.011 vs.0.017±0.006), the difference were statistical significant (all P<0.05). In addition, a significant increased expression of a-SMA was detected in the hepatitis B cirrhosis group (0.054±0.018), compared to normal control group (0.007±0.001), the difference was statistical significant, P<0.05. (4) The frequency of Th9 cells respectively had a positive correlation with prothrombin time and total bilirubin level (respectively:r=0.725, r=0.725), the difference had statistical significance (all P<0.05); the expression of a-SMA respectively had a positive correlation with the expression of CCR6 and CCL20 (respectively:r=0.492, r=0.516, all P<0.05); The levels of serum IL-9 was positively correlation with CCL20 level (r= 0.47, P< 0.05).Conclusion (1) Th9 cells and IL-9 may be involved in the occurrence and development of hepatitis B liver cirrhosis; (2) Th9 cells high expressed CCR4 and CCR6 in the microenvironment of hepatitis B cirrhosis; (3) CCL20/CCR6 chemokines axis may play a role during the process of Th9 cells recruitment. |
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