The Study Of NK Cells Function In Different Immune Phase Of Chronic Hepatitis B

Objective1、Study on the changes of NK cells function in different periods of chronic HBV infected patients.2、Horizontal analysis the differences in NK cells function under the different serum total bilirubin levels and correlation with ALT levels, or viral load, in HBV-related acute on chronic liver failure patients.3、Compared with NK cells frequency and functional changes in the patient’s course of treatment.Methods1、63cases of chronic HBV infection is divided into:(1) Patients in HBV-related acute on chronic liver failure(n=18), with the application of different nucleoside analogue antiviral treatment,87times extraction peripheral blood, by the lever of serum total bilirubin,divided into three groups that Tbil≥10ULN (upper limit of normal)(n=59),5ULN<Tbil<10ULN (n=17), Tbil<5ULN (n=11).(2)Chronic HBV infection patients in immune activation (n=30),without antiviral treatment.(3)Patients in immune tolerant phase with chronic HBV infection (n=15),observed changes in ALT. Healthy controls(n=32). These are tested no other history of hepatitis.2、Time-resolved fluorescence immunoassay quantitative techniques(TRFIA), quantitative detection of hepatitis B virus markers five indicators of hepatitis B surface antigen and antibody (HBsAg and HBsAb); e antigen and e antibody (the HBeAg and HBeAb); core antibody (HBcAb). Application Real time-PCR quantitative detection of HBV-DNA level. Abbott automatic biochemical analyzer liver function tests with nine indicators.3、Isolated peripheral blood lymphocytes (PBMCs) with Dakota human lymphocyte separation tube.4、Detected the frequency of of NK cells and its subtypes, with the expression levels of NK cell surface receptor (activating receptor/inhibitory receptor) by flow cytometry.5、The analysis of the level of IFN-γ and TNF-α secretion of NK cells by flow cytometry via intracellular cytokine staining method.6、Intracellular cytokine staining of granzyme B and perforin secreted by NK cells, this used to analysis of the cytotoxic activity of NK cells.7、Application Flowjo Software flow processing cytometry graph, SPSS20.0statistical analysis of the data, p-value<0.05was considered with significant difference.Results1、The analysis of the frequency and function of NK cells in different periods of Chronic HBV infection.(1) Compared with the healthy control group, the frequency of NK cells in the different periods of chronic HBV infection was significantly lower (P<0.05), the frequency of CD56dim NK cell subsets was lower than the healthy control group (P<0.001), and The frequency of CD56bright subsets higher than the healthy control group (P<0.001). The frequency of NK cells in patients with acute on chronic liver failure is lower than the IA group and the IT group (P<0.05).(2) Compared with the healthy control group, the activating receptor NKp46of NK cell in the IA group was significantly higher (P<0.05), the expression of NKp46, NKp30in the IT group also increased (P<0.05),whereas inhibitory receptor NKG2A expression IA group was decreased (P<0.05), the IT group had no significant difference. The expression of activating receptors and inhibitory receptor levels in ACLF group compared with healthy controls have no significantly different; among the groups compare activated and inhibitory receptors were not significantly different.(3) Compared with healthy controls, patients with chronic HBV infection secreting cytokines IFN-gamma were lower (P<0.001), compared to the cytokines TNF-alpha have no significant difference; secretion of IFN-yamong these groups have no significant differences, IA and IT group secretion of TNF-a level were higher than ACLF group, the difference was statistically significant (P<0.05).(4) Compared with healthy controls, patients with chronic HBV infection NK cells secreting granzyme B were higher, the difference of IA and ACLF group was statistically significant (P<0.05), all the groups secreting of perform were higher than the healthy control group (P<0.01). Among the groups compared to the IA group, the IT group level of secretion of granzyme B was significantly higher than the ACLF group, the difference was statistically significant (P<0.05); perforin among the groups did not differ significantly.2、Frequency and functional changes of NK cells in HBV-related ACLF patients with different levels of Tbil.(1) Patients with ACLF compared with healthy controls (P<0.01) showed lower NK cells in percentage of lymphocytes, frequency of the group CD56dimNK cell is lower than healthy controls (P<0.05),but frequency of CD56brightNK cell subsets is higher then healthy controls (P<0.05).(2) Compared with healthy controls, the activating receptor NKp46expression levels of NK cell in different lever of Tbil were not significant, Group1and Group3NKp30expression levels were higher (P<0.05), activating receptor NKG2D and inhibitory receptors NKG2A have difference. Compare activated receptor and inhibitory receptors were no significant differences among the groups in the different Tbil level. (3) Compared with healthy control, in the ACLF patients NK cells secreting IFN-y were significantly lower (P<0.01), no significant difference in TNF-α secretion; among the groups compare IFN-y and TNF-α,there was no significant difference.(4) The granzyme B level of ACLF patients show lower then healthy controls (P<0.01), but the secreting of perforin was higher than that in healthy controls (P<0.05), and group3was significantly higher than group2(P<0.05).3、Viral titer and ALT showed correlation with functional markers of NK cells in CHB patients.NK cells secrete cytokines IFN-gamma, and TNF-alpha was negatively correlated with ALT levels the chronic HBV infection patients. In ACLF patients, there have no significant correlation between the frequency of NK cells secrete cytokines IFN-γ, TNF-α and ALT lever. HBV-related ACLF patients NK cells secrete cytokines IFN-gamma, TNF-alpha was negatively correlated with HBV-DNA levels.4、The frequency and functional changes of NK cells in the treatment of patients with HBV-related ACLF. In during Tbil declining the frequency of NK cells and secretion of cytokines IFN-γ showed upward trend, but the difference was not statistically significant.cytotoxic activity of secretion perforin in NK cell showed downward trend.Conclusion1、Activity of NK cell function in different periods of chronic HBV infection have been various degree of inhibition compared with healthy controls, the patients with ACLF is restrained with higher degree.2、NK cells secrete cytokines IFN-gamma, and TNF-alpha was negatively correlated with ALT levels the chronic HBV infection patients. NK cell surface receptors NKD2A was positively correlated with ALT levels in ACLF patients, HBV-related ACLF patients NK cells secrete cytokines IFN-gamma, TNF-alpha was negatively correlated with HBV-DNA levels. 3、Dynamic observation ACLF patients with the recovery of the disease, the frequency of NK cells and secreting IFN-y increased trend,but cytotoxic activity show a downward trend.