HBV复制相关的肝细胞来源外泌体及其包裹的蛋白酶体调控单核细胞活化的机制研究

Hepatitis B virus (HBV) infection is a major health care problem worldwide. Exosomes secreted by different cell types in liver microenvironment may play important roles in HBV infection and pathogenesis. Our previous work has verified that HBV replication in liver cells could affect the protein component of exosomes produced by liver cells. Proteasome complex was enriched in HBV replicating liver cell derived exosomes. These exosomes could be enrolled by monocytes, activate monocytes and influence its IL-6 secretion level. The present study will further reveal the mechanism of monocyte activation modulated by exosomes derived from HBV-replicating liver cells. First, secretion levels of various cytokines produced by activated monocytes after treatment with exosomes produced by HBV-replicating liver cells will be evaluated. Then RNA-Seq transcriptomic and iTRAQ proteomic technologies will be used to analyze the modulating impact of HBV-replicating cell derived exosomes on monocytes activation in both gene and protein level. Stable knockdown of proteasome subunits in HBV-replicating cells will be established and their derived exosomes will be used to analyze the impact on monocyte activation. Further, the expression difference of these monocyte pathway genes will be verified in clinical samples. The current study will give an insight into the mechanism of monocyte activation induced by HBV-replicating liver cell produced exosomes, and provide a new perspective for revealing the mechanism of HBV infection and pathogenesis.

乙型肝炎病毒(HBV)感染严重危害人类健康。肝脏微环境中各种细胞来源外泌体可能在HBV感染和致病过程中发挥重要作用。我们前期工作证实HBV在肝细胞内复制会影响肝细胞外泌体的蛋白质组成，蛋白酶体复合物等在HBV复制肝细胞外泌体中富集，这些外泌体可被单核细胞吸收使其活化，并影响其分泌IL-6的水平。本研究将揭示HBV复制肝细胞来源外泌体对单核细胞活化的调控机制。首先分析HBV复制肝细胞来源的外泌体对单核细胞活化后产生各种细胞因子水平的影响，然后通过RNA-Seq转录组学和iTRAQ蛋白质组学技术探讨HBV复制肝细胞来源外泌体对被活化单核细胞的基因和蛋白质水平的调控，并构建稳定敲低蛋白酶体亚基的HBV复制细胞模型，分析其外泌体对单核细胞活化的影响，进一步在临床样本中验证这些基因的表达差异。本研究将为深入了解HBV复制肝细胞来源外泌体调控单核细胞活化的机制、阐明乙肝病毒感染与致病机理提供新视角。

乙型肝炎病毒(HBV)感染严重危害人类健康。肝脏微环境中各种细胞来源外泌体可能在HBV感染和致病过程中挥发重要作用。我们前期工作证实HBV在肝细胞内复制会影响肝细胞来源外泌体蛋白质组成，这些外泌体可被单核细胞吸收活化单核细胞，并影响其分泌IL-6的水平。本课题的研究拟进一步深入探讨HBV复制肝细胞来源外泌体对单核细胞活化的调控机制。首先，我们通过大量培养有或无HBV复制的HepAD38细胞，采用超速离心结合抗HBsAg的免疫共沉淀法分离纯化其外泌体，通过免疫印迹法和电子显微镜验证外泌体纯度。免疫印迹结果确认分离到的外泌体组分中可检测到外泌体标志蛋白质，电镜结果确认成功分离纯化了呈包膜囊泡特征结构的外泌体。随后，我们将获得的两组外泌体与人原代单核细胞共孵育，检测和分析HBV复制肝细胞来源外泌体对诱导单核细胞活化后分泌的各种细胞因子水平的影响，结果表明HBV复制细胞来源外泌体会影响单核细胞活化后分泌多种细胞因子包括IL-6、IL-12、IL-10和TNF-α的水平发生改变。进一步，本研究从转录组学和蛋白质组学多个层面，分析了HBV复制细胞来源外泌体对单核细胞活化后胞内基因和蛋白质表达的影响。通过RNA-seq转录组分析和非标记定量蛋白质组学分析技术，分别对HBV复制相关肝细胞来源外泌体与单核细胞共孵育24h或48h后的单核细胞，相比对照组PBS共孵育单核细胞分别进行转录组学和全细胞蛋白质组差异分析。结果显示，HBV复制肝细胞来源外泌体刺激单核细胞后，会引起单核细胞内多种生物学过程和信号通路发生改变，特别是与单核细胞趋化、迁移和粘附，以及细胞内信号传导等生物学过程的蛋白质和基因表达发生改变。本研究的结果，将为揭示乙肝复制细胞来源外泌体调控和活化单核细胞的分子机制提供新的线索，为进一步阐明乙肝病毒感染与致病机理提供新视角。