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Effects of IFN-α, Entecavir and Thymosin α1 on the Function of Dendritic Cells Derived from Chronic Hepatitis B Patients in Vitro

Author: LuGaoFeng
Tutor: TangZuoAi
School: Zhengzhou University
Course: Internal Medicine
Keywords: Chronic hepatitis B Dendritic cells Antigen-presenting Interferon α1 Entecavir Thymosin α1
CLC: R512.62
Type: Master's thesis
Year: 2007
Downloads: 178
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Abstract

The background and purpose of the hepatitis B virus (hepatitis B virus, HBV) infection is a global public health problem of hepatitis B endemic countries. Chronic hepatitis B (chronic hepatitis B, CHB) patients are often delayed healing, often by hepatitis develop cirrhosis, and even liver cancer, immune reactions play an important role in this process. Studies have shown that one of the important mechanisms of hepatitis B chronic HBV antigen-specific T-cell immune tolerance, patients can not produce the immune response against HBV-specific cytotoxic T lymphocytes (cytotoxic T lymphocyte, CTL), so can not be rid of the virus. One of the important reasons causing the CHB immune tolerance dendritic cells (dendritic cell, DC) defects, virus antigen signal can not be effectively passed to the immune system of the patient's body. Therefore, the number of recovery DC or enhance DC antigen-presenting function, full activation of the immune response has become an important way of treatment of CHB. DC is able to activate to initialize T lymphocytes important antigen presenting cells, play an important role in the anti-viral cell immunity. Drop-stimulating factor (GM-CSF) and interleukin -4 (IL-4) induced in vitro by granulocyte - macrophage amplification can get enough peripheral blood monocyte-derived DC (monocyte-derived DC of MoDC) . CD1a expression in human thymus cells and DC, human DC relative to the characteristic logo, CD83 is an important marker of mature DC, CD80, HLA-DR surface costimulatory molecules. Interferon α (interferon-α, IFN-α) and nucleoside (acid) analogues is recognized at home and abroad with chronic hepatitis B antiviral therapy drug. In addition to direct inhibition of HBV replication, IFN-α, is also an important way to enhance the body's immunity to HBV, its mechanism is more complex; grace entecavir (entecavir, ETV) is a novel oral nucleoside drugs, by phosphorylation of intracellular after, and the synthesis of deoxycytidine (dCTP) competition into the virus DNA chain termination of viral DNA replication. Thymosin α1 enhance non-specific immune function, used for joint treatment of CHB. But the impact of these drugs for patients with CHB DC, less reported in the literature, especially ETV as CHB antiviral clinical strongest, the immune system not been reported. In this study, IFN-α, ETV and thymosin α1 MoDC in vitro with CHB peripheral blood were incubated culture fluid IL-6 by measuring the expression of the phenotype the DC surface CD1a, CD83, CD80, HLA-DR molecules detected DC observe their impact the DC biological characteristics of CHB, IL-12 content and allogeneic mixed lymphocyte reaction, a series of indicators for the clinical application of these drugs and their DC vaccine combined treatment of CHB find experimental evidence. Materials and Methods acquisition CHB patients and healthy human peripheral fresh venous blood heparin through lymphocyte separation liquid outer density gradient centrifugation to obtain mononuclear cells with the 10% FBS-containing RPMI 1640 medium, resuspend the mononuclear cells inoculated into 24-well cell culture plate after standing for 2 hours, gently eluted nonadherent suspension cells, adherent cells with complete medium (RPMI 1640 containing 10% fetal bovine serum containing 10ng/ml rhGM-CSF and for Cd is 5ng / ml rhIL-4) induced amplification, 37 ° C, 5% CO ₂ humidified incubator culture. The next day, half the amount was changed. Collected five days in DC, the the CHB source DC were added to a certain concentration of IFN-α (300U/ml), thymosin α1 (0.1μg/ml) and ETV (0.05μg/ml) divided into IFN-α group thymosin α1 group ETV group and IFN-α thymosin α1 group, set healthy control group and CHB control group at the same time, continue to co-culture, and eight days harvest each group DC following correlation detection: 1. Dynamic Morphological changes were observed under an inverted microscope. 2. Determination of flow cytometry (FCM) DC phenotype molecules of CD1a, CD83, CD80 and HLA-DR expression. 3. Allogeneic mixed lymphocyte reaction method for the determination of DC to stimulate lymphocyte proliferation. 4. ELISA assay DC culture supernatant IL-12, IL-6 content. Results 1. Culture 8d observe the cells in each group, and the healthy control group floating in the culture medium in the more nebulous DC cell mass, the surface protrusions rich; drug treatment group were visible surface protrusions rich DC; CHB control group DC irregular shaped , polygonal; CHB healthy control group and each-CHB drug treatment group cell differentiation morphology than the control group. 2. Section 8d of culture groups DC phenotype molecules CD1a, CD80, CD83 and HLA-DR expression, CHB control group and CHB drug treatment group (P <0.05) was significantly higher than in the healthy control group; CHB control group lower than The CHB drug treatment group (P <0.05); thymosin α1 IFN-α group, IFN-α group and thymosin α1 group no significant difference (P> 0.05), but were higher than the ETV group (P <0.05). 3. Healthy control group DC to stimulate allogeneic T cell proliferation strongest; CHB control group DC to stimulate allogeneic T cell proliferation weakest; each CHB drug treatment group strong in CHB control group; IFN-α thymosin α1 group IFN-α group and thymosin α1 group no significant difference (P> 0.05), but higher than the ETV group (P <0.05). 4. Each group DC culture supernatant of IL-12 content of the highest for the healthy control group, followed by the drug-treated group CHB, CHB control group the lowest (P <0.05); IL-6 content contrast (P <0.05). Conclusion 1. RhGM-CSF and rhIL-4 induced by the combined method can successfully from peripheral blood mononuclear cells induced in vitro to differentiate into DC, to provide a sufficient amount of cells for further experimental research. 2. CHB's DC poorly differentiated membrane surface molecules of CD1a, CD80, CD83, and HLA-DR expression decreased stimulate lymphocyte proliferation and secretion of IL-12 is low, suggesting functional defects compared with healthy people may HBV one of the important causes of chronic infection. 3. IFN-α, ETV and thymosin α1 can be improved in different degrees of CHB DC surface molecules of CD1a, CD83, CD80, HLA-DR expression and promote their secretion of cytokines IL-12 and enhance DC stimulate lymphocyte proliferation capacity, enhance cellular immune function. 4. Promote DC function, ETV than IFN-alpha and thymosin α1 weak; IFN-α and thymosin α1 with IFN-α or thymosin α1 difference was not statistically significant.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis B
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