| Purpose: To investigate the correlation of androgen receptor (AR) and estrogen receptor a (ERa) in peripheral blood mononuclear cells (PBMCs) and hepatic tissue with the histopathology status of hepatic tissue, hepatitis B virus (HBV) replication and antigen expression, CD4+CD25+ regulatory T cells (Treg) and T helper cells 17 (Th17) in patient with chronic hepatitis B (CHB).Materials and methods:1 The subject investigated1.1 Inclusion criteria:With reference to the diagnostic criteria of the"Guideline on prevention and treatment of CHB in China (2010)", we selected 94 cases of CHB patients with liver biopsy, without gender and age limitation;1.2 Exclusion criteria:1) With therapy of anti-virus drug; 2) Alcoholic liver disease, non-alcoholic fatty liver disease, hepatitis C, drug-induced hepatitis, autoimmune liver disease, cholestatic liver disease, congestive liver disease, liver degeneration, hemochromatosis and other non-hepatitis B virus infection caused by liver disease; 3) Patients who had used hormone replacement drugs, hormone antagonist drugs, licorice acid drugs,or antifibroticin drugs the nearly six months; 4)Patients who diagnosis decompensated liver disease; 5) Patients mixed with gonadal disease, endocrine diseases or diseases that may affect the body testosterone and estradiol level.2 Acquisition of the experimental specimens2.1 Acquisition of PBMCs:PBMCs obtained by means of Ficoll density gradient centrifugation is divided into two parts. One is added with 300μl RNAlater,the other one with freezing medium. Freeze the cells in minus 80 degrees refrigerator.2.2 Acquisition of the liver tissue:We used one second percutaneous transhepatic method to extract liver tissue at length of longer than 1.2cm which is divided into two parts.One is longer than 1.0cm which is for pathological examination, the other one longer than 0.2cm is preserved with RNAlater in minus 80 degrees refrigerator.3 Method of detecting the relevant indicators3.1 Frequency of Th17 cell and Treg cell in PBMCs:Detect the frequency of Th17 (CD3+CD8-IL17+) and Treg (CD3+CD8-CD25+CD127-) cell in PBMCs with flow cytometry.3.2 Quantity of AR mRNA, ERa mRNA, Foxp3 mRNA, RORyt mRNA in PBMCs and liver:Detect with RT-PCR.3.3 Quantity of Foxp3+T cell and IL-17+T cell in liver:Measure with IHC. Then pick five portal areas randomly and count average quantity of Foxp3+T cell and IL-17+Tcell.3.4 The liver pathological diagnosis:After HE staining and masson staining, The liver tissue pathological G and S were determined after reading by authority pathologists.3.5 HBsAg and HBcAg expression of liver tissue were measured by IHC;3.6 Quantity of HBV markers in blood serum:With the chemiluminescent micro-particle immunoassay.3.7 Quantity of HBV-DNA in blood serum:With RT-PCR.Results:1 For HBeAg positive patients, the quantity of ERa mRNA in PBMCs have a positive correlation with the liver pathological G state (r=0.303, P=0.034). The quantity of AR mRNA in liver have a negative correlation with the liver pathological G and S state (r=-0.432, P=0.002; r=-0.353, P=0.014). For HBeAg negative patients, the quantity of AR mRNA in PBMCs have a negative correlation with the liver pathological G and S state (r=-0.362, P=0.019; r=-0.346, P=0.025).The quantity of AR mRNA in liver have a negative correlation with the liver pathological G and S state (r=-0.592, P=0.000; r=-0.472, P=0.001)2 For HBeAg positive patients and HBeAg negative patients, the quantity of AR mRNA and ERa mRNA in PBMCs and liver have no correlation with the quantity of HBsAg and HBcAg in liver, HBsAg in blood serum and HBV DNA(P>0.05).3 For HBeAg positive patients, the quantity of ERa mRNA in PBMCs have a positive correlation with the quantity of Foxp3 mRNA、RORyt mRNA in PBMCs (r=0.557, P=0.000; r=0.610, P=0.000). The quantity of ERa mRNA in liver have a positive correlation with the quantity of RORyt mRNA、RORyt mRNA/Foxp3 mRNA in liver (r=0.550, P=0.000; r=0.485, P=0.000). For HBeAg negative patients, the quantity of ERa mRNA in PBMCs have a positive correlation with the quantity of Foxp3mRNA、RORyt mRNA in PBMCs (r=0.478, P=0.001;r=0.585, P=0.000). The quantity of AR mRNA in liver have a negative correlation with the quantity of RORyt mRNA in liver (r=-0.371, P=0.013). The quantity of ERa mRNA in liver have a negative correlation with the quantity of RORyt mRNA, RORyt mRNA/Foxp3 mRNA in PBMCs (r=-0.509, P=0.001; r=-0.336, P=0.030) and have a positive correlation with the quantity of RORyt mRNA in liver (r=0.407, P=0.006)4 For HBeAg positive patients, the quantity of Foxp3 mRNA in PBMCs have a positive correlation with the liver pathological G and S state (r=0.448, P=0.001; r=0.322, P=0.024). The quantity of RORyt mRNA in PBMCs have a positive correlation with the liver pathological G state(r=0.342, P=0.016). The quantity of RORyt mRNA/Foxp3 mRNA in liver have a negative correlation with the liver pathological G and S state (r=-0.649, P=0.000; r=-0.568, P=0.000).For HBeAg negative patients, the quantity of Foxp3 mRNA in liver have a positive correlation with the liver pathological G state (r=0.348, P=0.021). The quantity of RORyt mRNA in liver have a positive correlation with the liver pathological S state (r=0.410, P=0.006; r=0.381, P=0.011).Conclusion:1 Mechanism for AR and ERa affecting pathogenesis and progress of CHB may be different between HBeAg positive patients and negative patients. The decrease of AR mRNA in liver may be an independently dangerous factor which influences the pathogenesis and progress of CHB both for HBeAg positive patients and negative patients. Treg cells and Th17 cells may also be closely related with the pathogenesis and progress of CHB.2 Mechanism for AR and ERa affecting pathogenesis and progress of CHB may be does not depend on influencing on the duplication of HBV and antigen expression and may be depend on affecting activation and generation of Treg cells and Th 17 cells. |
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