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白细胞介素-21参与慢性丙型肝炎或乙肝肝硬化免疫发病机制研究
冯国华
2013-11-18
导师王福生
专业内科学
授予单位北京大学
授予地点北京大学解放军302医院教学医院
学位博士
关键词白细胞介素-21,慢性丙型肝炎,乙肝肝硬化,病毒特异性CD8+ T细胞,NK细胞,肝星状细胞
其他题名Immune pathogenesis mediated by Interleukin-21 in chronic hepatitis C or HBV-associated liver cirrhosis patients
分类号R512.6
摘要目的:分别研究白细胞介素-21(IL-21)对慢性丙型肝炎患者体内病毒特异性CD8+ T细胞和NK细胞的免疫调节作用以及在慢性乙肝肝硬化(HBV-LC)发病机制中的作用。方法:在本研究中,我们使用流式细胞仪分别检测了慢性丙型肝炎患者外周血中病毒特异性IL-21+CD4+ T细胞频率;加入IL-21前后,病毒特异性CD8+ T细胞的频率及病毒特异性CD8+ T细胞中CD107a,颗粒酶A(GrA),颗粒酶B(GrB),穿孔素(Perforin),Ki-67的表达。使用流式细胞仪观察了NK细胞和K562细胞共培养后,7-AAD阳性细胞的频率和NK细胞中GrB,Perforin,干扰素-γ(IFN-γ)和CD107a等的表达。检测了使用佛波酯(PMA)和离子霉素刺激后乙肝肝硬化患者和健康人外周血中IL-21+CD4+ T细胞的频率;血浆中IL-21的水平;同时使用免疫组化的方法观察了IL-21+细胞在肝脏中的分布;最后观察了IL-21对肝星状细胞体外细胞系LX-2活化、增殖及凋亡的影响。结果:我们发现慢性丙型肝炎患者外周血中病毒特异性IL-21+CD4+ T细胞频率与HCVRNA呈明显负相关,提示可能与病毒清除有关;加入IL-21后,可明显提高慢性丙型肝炎患者病毒特异性CD8+ T细胞频率及CD107a,GrB,Perforin,Ki-67的表达。同时,我们还发现慢性丙型肝炎患者外周血中NK细胞表面白细胞介素-21受体(IL-21R)较健康人明显升高;加入IL-21后,可明显提高慢性丙型肝炎患者和健康人外周血中NK细胞杀伤K562细胞的能力,同时可上调杀伤过程中NK细胞GrB,Perforin,IFN-γ和CD107a的表达。最后,我们还发现HBV-LC患者外周血及肝脏中IL-21的含量明显升高;IL-21可在体外促进LX-2细胞的活化,抑制LX-2细胞的凋亡及促进胶原分泌。结论:本研究结果提示IL-21可增强慢性丙型肝炎患者外周血中病毒特异性CD8+ T细胞及NK细胞的杀伤功能,同时还可以促进病毒特异性CD8+ T细胞的增殖,并且在HBV-LC纤维化形成过程中有一定的促进作用。
英文摘要Objective: To investigate the immune regulation of IL-21 on HCV-specific CD8 T cells and NK cells in chronic hepatitis C and its role in HBV-associated liver cirrhosis. Methods: The peripheral frequency of HCV-specific IL-21+ CD4+ T cells in chronic hepatitis C patients was detected by flow cytometry; the frequency of HCV-specific CD8+ T cell before and after the administration of IL-21 was compared. Meanwhile, the expression of CD107a, GrB, Perforin and Ki-67 in HCV-specific CD8+ T cells were analysed. Then the frequency of 7AAD-positive cells and GrB, Perforin, IFN-γ and CD107a expression in NK cells were detected by flow cytometry after NK cells and K562 cells were co-cultured. In addition, the frequency of IL-21+ CD4+ T cell in peripheral blood stimulated by PMA and ionomycin was analysed by flow cytometry in patients with HBV-associated liver cirrhosis and healthy controls. The plasma level of IL-21 was measured by ELISA and the in situ distribution of IL-21+ cells in liver was observed by immunohistochemistry. Finally, the activation, apoptosis and proliferation of LX-2 cells in vitro were examined. Results: The frequency of HCV-specific IL-21+ CD4+ T-cell in peripheral blood was significantly negatively correlated with HCVRNA in patients with chronic hepatitis C, suggesting that HCV-specific IL-21+ CD4+ T-cell might be related to viral clearance; the frequency of HCV-specific CD8 + T cell and the expression of CD107a, GrB, Perforin and Ki-67 were significantly increase by the administration of IL-21. At the same time, we found that peripheral expression of IL-21R on NK cells in patients with chronic hepatitis C increased significantly when compared with healthy controls; Cytotoxicity of NK cells targeting K562 cells were significantly improved by the administration of IL-21 in both patients with chronic hepatitis C and healthy controls; Meanwhile, administration of IL-21 also increased GrB, Perforin, IFN-γ and CD107a expression in NK cells. Finally, level of IL-21 was significantly increased in peripheral blood and liver of patients with HBV-associated liver cirrhosis; In vitro administration of IL-21 was accompanied with activating LX-2 cells, inhibiting LX-2 cells apoptosis, and up-regulating collagen production by LX-2 cells. Conclusion: The data in this study indicate that IL-21 could increase cytotoxicity of HCV-specific CD8+ T cells and NK cells in peripheral blood of patients with chronic hepatitis C, and promote the proliferation of virus-specific CD8+ T cells. In addition, IL-21 may contribute to the fibrogenesis of HBV-associated liver cirrhosis.
语种中文
出处http://xuewei.bjmu.edu.cn/simpsearch.action?keyword=白细胞介素-21参与慢性丙型肝炎或乙肝肝硬化免疫发病机制研究&dbid=72
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/105426
专题北京大学解放军302医院教学医院
作者单位北京大学解放军302医院教学医院
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冯国华. 白细胞介素-21参与慢性丙型肝炎或乙肝肝硬化免疫发病机制研究[D]. 北京大学解放军302医院教学医院. 北京大学,2013.
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