| Research backgroundHepatitis B virus(HBV)is a hepatotropic DNA virus that has infected 350 million people globally.HBV-infection-associated liver disease is a series of diseases,including chronic hepatitis B(CHB),liver cirrhosis(LC),Hepatocellular Carcinoma(HCC),et al.It has been seriously threatening human's health because of the character of high incidence,long course of the disease,and poor prognosis.In the past,it was widely considered that continuous HBV replication was the pathological basis of HBV-infection-associated liver disease,leading to hepatic cell damage and liver function damage.So,the treatment of HBV-infection-associated liver disease is mainly focused on antiviral therapy.However,the treatment of antiviral therapy has some limitations and drawbacks.On the one hand,side effects during treatment,such as virus mutations and drug resistance,seriously affect the efficacy of the drugs;on the other hand,treatment of antiviral therapy can not completely alleviate the progression of HBV-infection-associated liver disease.Therefore,exploration of new pathogenic mechanism of HBV-infection-associated liver disease to control the progression of HBV-infection-associated liver disease and find better therapy is a new direction of current research.The latest research shows that,during the progression of HBV-infection-associated liver disease,the interaction of cellular immune response and virus is more important than that of hepatitis B virus itself,with T lymphocytes playing a vital role in the immune response to persistent HBV infection.Therefore,it is especially important to investigate the relationship between the T cell-mediated immune response and the pathogenic mechanism of HBV-infection-associated liver disease.Treg and Th17 cells are derived from the same original T cells.A close,complex relationship exists between Treg and Th17 cells.Although most of the chemotactic receptors on the surface of these two T cell subsets are the same,their functions are antagonistic to each other.Treg cells secrete immunosuppressive cytokines to sup-press the immune response,while Th17 cells can increase the immune response by releasing inflammatory cytokines.Under normal circumstances,Treg and Th17 cells maintain a dynamic balance and result in the induction of immune responses of appropriate intensity,which is conducive to the maintenance of a stable immune state in the body.Treg/Thl7 imbalance will cause abnormal immune response and it is closely related to many immune disorders,tumors,and infectious diseases.Latest opinions show that Treg/Th17 imbalance maybe take part in the mechanism ofHBV-infection-associated liver disease.Current studies have confirmed that Treg/Thl7 imbalance is present in patients with chronic hepatitis B.In addition to inhibiting an excessive immune response and alleviating liver inflammation,Tregs and related cytokines also indirectly lead to chronicity of viral infection.Th17 participate in specific immune responses to HBV that promote inflammation and have potential antiviral effects.Therefore,Treg/Th17 imbalance plays a vital role in the mechanism of chronic hepatitis B.However,these changes have not been studied systematically in LC and HCC patients.What are the changes of Treg and Thl 7 cells during the progression of Hepatitis B-associated liver cirrhosis and HBV-related liver cancer?Does an imbalance in Treg/Thl7 cells exist in peripheral blood of patients with hepatitis B-associated liver cirrhosis and HBV-related liver cancer?What is the relationship between the Treg/Th17 imbalance and the mechanism of hepatitis B-associated liver cirrhosis and HBV-related liver cancer?These are the purpose of this research.Part 1Treg/Th17 Imbalance and Its Clinical Significance in Patients with Hepatitis B-associated Liver CirrhosisObjective1.To insure if Treg/Th17 imbalance exists in peripheral blood of patients with hepatitis B-associated liver cirrhosis.2.To detect the relationship between Treg and Th17 and Treg/Th17 ratio with clinical stages of hepatitis B-associated liver cirrhosis.3.To detect the correlation between Treg and Th17 in patients with hepatitis B-associated liver cirrhosis.MethodsA total of 93 patients with hepatitis B-associated liver cirrhosis,who had never been treated before seeking treatment from the Department of Hepatology,Qilu Hospital of Shandong University,between January 2016 and July 2016,were selected as the liver cirrhosis(LC)group.According to the Child-Pugh classification,45 patients were classified into the compensated liver cirrhosis(CLC)group,and 48 patients were classified into the decompensated liver cirrhosis(DCLC)group.A total of 40 healthy subjects from hospital check-center were enrolled into the control(Ctrl)group.Treg and Th17 cells in peripheral blood of study subjects were detected by flow cytometry.Results1.Compared with the Ctrl group,the LC group was found to have a significantly reduced percentage of CD4+CD25+FOXP3+Treg cells(P<0.01),markedly increased CD4+IL-17+Thl7 cells(P<0.001),and a significantly lower Treg/Thl7 ratio(P<0.001).2.Compared to the Ctrl group,both the CLC group(P<0.01)and the DCLC group(P<0.001)showed lower percentages of CD4+CD25+FOXP3+Treg cells,with the percentage of CD4+CD25+FOXP3+Treg cells of the DCLC group being lower than that of the CLC group(P<0.001).Compared to the Ctrl group,both the CLC group(P<0.001)and the DCLC group(P<0.001)showed higher percentages of CD4+IL-17+Th17 cells,with the percentage of CD4+IL-17+Thl7 cells of the DCLC group being higher than that of the CLC group(P<0.001).Compared to Ctrl group,both the CLC group(P<0.001)and the DCLC group(P<0.001)showed decreased Treg/Th17 ratios,with the Treg/Th17 ratio of the DCLC group being lower than that of the CLC group(P<0.001).3.The Spearman correlation analysis showed a negative correlation between the Treg and Th17 cells in the LC group(r=-0.6386,P<0.0001).ConclusionTreg/Th17 imbalance exists in patients with hepatitis B-associated liver cirrhosis,with reduced Treg cells in their peripheral blood,increased Th17 cells and decreased Treg/Th17 ratio.Treg and Th17 cells are negatively correlated.Treg and Th17 cells and Treg/Th17 ratio are closely related to the clinical stage of hepatitis B-associated liver cirrhosis.These results indicate that Treg/Th17 imbalance not only participates in the pathogenesis of hepatitis B-associated liver cirrhosis,but also has a close relation with the progression and prognosis of hepatitis B-associated liver cirrhosis.Further research of Treg/Th17 imbalance maybe offer a new therapeutic target for the treatment of hepatitis B-associated liver cirrhosis.Part 2Treg/Th17 Imbalance and Its Clinical Significance in Patients with HBV-related Liver CancerObjective1.To insure if Treg/Thl7 imbalance exists in peripheral blood of patients with HBV-related liver cancer.2.To detect the relationship between Treg and Th17 and Treg/Th17 ratio with clinical stage,tumor size of HBV-related liver cancer.3.To detect the correlation between Treg and Th17 in patients with HBV-related liver cancer.MethodsA total of 51 patients with HBV-related liver cancer,who had never been treated before seeking treatment,from the Department of Hepatology,Qilu Hospital of Shandong University,from May 2013 to March 2016,were selected as the HCC group.According to the tumour-node-metastasis(TNM)classification and tumor size,35 patients were classified into the early(stage ?-?)HCC group and 16 patients were classified into in the advanced(stage ?-?)HCC group;35 patients were classified into a small tumour size(size<5cm)group and 16 patients were classified into in the large tumour size(size?5cm)group.Forty-two healthy individuals from hospital check-center were enrolled as the control group.Treg and Th17 cells in peripheral blood of study subjects were detected by flow cytometry.Results1.The proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the HCC group than in the control group(P<0.001).The proportion of CD4+IL-17+Th17 cells was remarkably higher in the HCC group than in the control group(P<0.001).The Treg/Th17 ratio was also remarkably higher in the HCC group tlhan in the control group(P<0.001).2.The proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the different HCC tumour stage groups than in the control group(P<0.001);moreover,the proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the stage?-? group than in the stage ?-? group(P<0.001).The proportion of CD4+IL-17+Th17 cells was significantly higher in the different HCC tumour stage groups than in the control group(P<0.001);moreover,the proportion of CD4+IL-17+Th17 cells was significantly higher in the stage ?-? group than in the stage ?-? group(P<0.001).The Treg/Th17 ratio was significantly higher in the different HCC tumour stage groups than in the control group(P<0.001);however,there was no difference in the Treg/Thl7 ratio in the stage ?-? group and the stage ?-? group(P>0.05).3.The proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the HCC groups with various tumour sizes at early stages ?-? than in the control group(P<0.001);moreover,the proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the size?5cm group than in the size<5cm group(P<0.001).The proportion of CD4+IL-17+Thl7 cells was significantly higher in the HCC groups with various tumour sizes at early stages ?-? than in the control group(P<0.01);moreover,the proportion of CD4+IL-17+Thl7 cells was significantly higher in the size>5cm group than in the size<5cm group(P<0.05).The Treg/Th17 ratio was significantly higher in the HCC groups with different tumour sizes at early stages ?-? than-in the control group(P<0.05);however,there was no difference in the Treg/Th17 ratio between the size?5cm group and the size<5cm group(P>0.05).The proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the HCC groups with various tumour sizes at an advanced stage III-IV than in the control group(P<0.001);moreover,the proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the size?5cm group than in the size<5cm group(P<0.01).The proportion of CD4+IL-17+Th17 cells was significantly higher in the HCC groups with various tumour sizes at an advanced stage ?-? than in the control group(P<0.05);moreover,the proportion of CD4+IL-17+Th17 cells was significantly higher in the size>5cm group than in the size<5cm group(P<0.05).The Treg/Th17 ratio was significantly higher in the HCC groups with different tumour sizes at an advanced stage ?-? than in the control group(P<0.001);however,there was no difference in the Treg/Th17 ratio between the size>5 cm group and the size<5cm group(P>0.05).The proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the HCC groups with various tumour sizes than in the control group(P<0.001);moreover,the proportion of CD4+CD25+FOXP3+Treg cells was significantly higher in the size>5 cm group than in the size<5cm group(P<0.001).The proportion of CD4+IL-17+Th17 cells was significantly higher in the HCC groups with various tumour sizes than in the control group(P<0.001);moreover,the proportion of CD4+IL-17+Th17 cells was significantly higher in the size>5cm group than in the size<5cm group(P<0.001).The Treg/Thl7 ratio was significantly higher in the HCC groups with different tumour sizes than in the control group(P<0.01);however,there was no difference in the Treg/Th17 ratio between the size>5cm group and the size<5cm group(P>0.05).4.The Spearman correlation analysis showed a positive correlation between the Treg and Th17 cells in the HCC group(r=0.4895,P<0.001).ConclusionTreg/Th17 imbalance exists in patients with HBV-related liver cancer,with increased Tregs in their peripheral blood,higher Th17 cells and increased Treg/Th17 ratio.Treg and Th17 cells are positively correlated.Both of them are closely related to the clinical stage and tumor size of HBV-related liver cancer.These results indicate that Treg and Th17 cells not only participate in the pathogenesis of HBV-related liver cancer,but also have a close relation with the progression and prognosis of HBV-related liver cancer.Regulation of the differentiation of Treg and Th17 cells and correction of the Treg/Th17 imbalance are important to control the progression of HBV-related liver cancer and improve the clinical outcome.Further study of Treg/Th17 imbalance may provide a new way for immunotherapy of HBV-related liver cancer. |
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