| In this thesis, we focused on the screening index of Polyethylene glycol (PEG) interferon treatment of chronic hepatitis B and the role of NK cells acted in chronic hepatitis B infection. This thesis consists of two sections.In the first section, we were trying to screen the index which could guide the PEG interferon treatment of chronic hepatitis B. Hepatitis B virus (HBV) infection is one of the most critical public health issues which threatened the human health in our country, even in the world. Recent therapy methods could be divided into two categories, interferon (IFNα, PEGIFNα) and nucleotide analogs (lamivudine、 Telbivudine, etc.). Due to the dual role (direct antiviral and immune regulation) interferon acted in the therapy process, it has been widely applied in clinical antiviral treatment. The interferon has numerous advantages including no drug resistance, immune-mediated control HBV infection. All of these features offered more opportunities to obtain higher HBeAg seroconversion, longer virological response and better HBsAg clearance, which could help the patients maintaining the HBV DNA content under the detection limit. However, the treatment effect rate of interferon-a based conventional method is ranging from25%to40%, the recurrence rate and subsequent effect after drug withdrawal rate is ranging from15%to20%and the persistent effect rate is only25%～40%. Moreover, the treatment effect was largely related to the individual difference, more than30%of the cases require long time treatment, generally, longer than six months. In addition, from the aspects of treatment costs, interferon-a is very different from other drugs, the average cost of PEGIFNa-2a/2b is much higher than that of LAM, ADV and ETV drugs. For instance, chronic hepatitis B patients utilize interferon-a based therapy for more than six months, even twenty four months antiviral treatment, the failure of treatment will cause not only physical and mental suffering, but also a tremendous economic pressure. Though there are some indicators used for interferon-a response prediction, it does not meet the clinical needs. To address this issue, we firstly gathered the serum samples from chronic hepatitis B patients before and after twenty four weeks polyethylene glycol interferon treatment. Then, categorizing them into two groups based on the virologic and non-virological response. Utilizing luminex200detects forty six kinds of human cytokines, all the samples’CXCL9and IP-10baseline virological response group higher than the non-virological response group and virological group fell in the process of treatment, whereas non-virological response group was no significant change. Virological response group compared with the non-virological response, MIP-ld at baseline and during treatment were significantly higher than non-virological response, while TARC at baseline and during treatment were significantly lower than in non-virological response. HBV DNA, HB eAg, TARC, MIP1d, CXCL9, CXCL6and IP10baseline level has an expected effect for interferon treatment. The AUROC value of HBV DNA, HB eAg, TARC, MIP1d, CXCL9, CXCL6and IP10were0.879,0.964,0.77,0.787,0.799,0.722and0.787(p=0.001, p<0.001, p=0.021, p=0.01, p=0.01, p=0.05and p=0.013). Additionally, CXCL9, CXCL6and ALT have obvious correlation(r=0.588, p=0.002; r=0.530, p=0.005). In summary, we found that cytokines level of pre-treatment and in-treatment has the potential to select chronic hepatitis B patients who can respond to PEG-IFN. Therefore, cytokines can be used as an index for former choice drug therapy of chronic hepatitis B.The second section was an investigation about the antiviral immunity reaction mechanism of NK cells in the process of chronic hepatitis b virus infection. Patients with chronic HBV infection are lacking of effective antiviral immunity reaction. The host cannot remove the virus timely and effectively, resulting in persistent infection. This immune tolerance state may due to the host anti HBV cellular immune responses in a state of incompetence or suppressed. Latest research has shown that the activation of NK cells suppresses T cell responses is responsible for this immune tolerance state formation in chronic HBV infection. NK cells use different ways to limit T cell function, including antigen affecting induced function change, cytokine secretion altered reaction, lytic activity mediated immunity reaction and etc. Depletion of NK cells or NK-deficient mice demonstrates that NK cells have an intrinsic ability to inhibit adaptive T cell responses against virus, it becomes one of the most promising directions for developing new immunotherapy drugs of chronic HBV. We investigated the mechanism of the role which NK cells acted in chronic hepatitis B both in vivo and in vitro. Selective depletion of NK cells using25μg anti-NK1.1monoclonal antibodies in HBV transfecting mice resulted in serum HBV DNA and HbsAg levels decreased significantly, the most significant change occurs in the deletion of NK cells at the third day. Comparing with pre-injection, in serum HBV DNA decreased1.51og10, and HBsAg decreased1.0log10. In vitro, first of all, extracting PBMC from patients with chronic hepatitis B and sorting delete NK cells, followed by using the core antigen peptide libraries co-cultured with PBMC and NK cells deleted PBMC. The results showed that the majority of patients can recover virus-specific cell proportional of CD8+IFN-γ+T cells after NK deletion. To sum up, we have preliminary confirmed that the deletion of NK cells could restore the host state of antiviral and laid a foundation for chronic hepatitis B target as a novel immunotherapy. |
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