慢性乙型肝炎患者妊娠期NK细胞亚群变化及其调控Th17细胞介导的炎性反应机制研究

Hepatitis B during pregnancy is one of the infectious diseases with high incidence, however the pathogenesis of aggravating hepatitis B remains unclear. During acute or chronic viral infection, NK cells not only participate in early antiviral immunity, but also a growing number of studies suggest that the regulation of the NK cells involved in adaptive immunity. Our previous work had shown that the percentage of CD56brightCD16- NK cells was significantly lower in chronic hepatitis B patients during pregnancy. IFN-γ expression of NK cells was significantly downregulation with elevated levels of serum IL-17. Moreover, the percentage of CD56brightCD16- NK cells showed a negative correlation with the level of serum IL-17. Thus, we hypothesize the diminishing regulation of CD56brightCD16- NK cells on Th17 cells maybe associated with the aggravating hepatitis B during pregnancy. Therefore, the aim of this present project is to define the regulatory role of NK cells on Th17 cells by analysis of healthy pregnant women, HBV-carrier pregnant women and pregnant women with chronic hepatitis B, including the NK cells phenotype and function as well as the proportion and function of Th17 cells. We sought to find out the cytokine that inhibit the polarization of Th17 cells. We will clarify the mechanisms using co-culture assay, experiments with the blocking antibody and HBsAg transgenic pregnant mouse model. In conclusion, this project will provide the experimental basis and an important sight for prevention and treatment of chronic hepatitis B during pregnancy.

妊娠期乙型肝炎是妊娠期感染性疾病中的高发疾病，其加重机制并不清楚。在急性或慢性病毒感染过程中，NK细胞不仅参与早期抗病毒免疫，而且近期研究提示NK细胞也参与对适应性免疫的调节。我们前期结果表明乙型肝炎发病期孕妇外周血中CD56brightCD16-NK细胞亚群减少，其IFN-γ分泌水平降低，血浆IL-17水平升高。CD56brightCD16-NK细胞比例和血浆IL-17水平呈负相关。我们提出假设，妊娠期乙型肝炎的加重可能与CD56brightCD16-NK细胞对Th17细胞的调控减弱有关。本课题拟全面比较乙型肝炎孕妇、HBV携带期孕妇及健康孕妇NK细胞的表型和功能以及Th17的比例和功能差异，并探究其机制，阐明NK细胞影响Th17细胞的极化过程的关键分子；通过细胞共培养实验、抗体阻断实验以及HBsAg转基因妊娠小鼠模型进行验证。最终为妊娠期乙型肝炎的预防和治疗提供实验基础和理论依据。

NK细胞在妊娠期乙型肝炎中作用研究尚不清楚。在本课题研究中，我们发现妊娠期HBeAg阳性更易出现肝炎加重患者外周血中CD56brightCD16-NK细胞比例显著降低，而CD56dimCD16+NK比例显著增加。进一步对NK细胞的表型、细胞因子分泌功能研究发现，妊娠期HBeAg阳性乙型肝炎患者外周血NK细胞与杀伤功能相关的活化性受体CD226和NKG2D的表达显著上调，抑制性受体NKG2A和CD158b的表达显著下调，且与肝功能和HBV病毒载量有关联。HBeAg阳性孕妇NK细胞亚群发生改变的同时其分泌Th1型细胞因子能力也降低，表现为IFN-γ表达水平降低，而Th17细胞数量明显增加。最后，通过体外抗体阻断实验和细胞共培养实验，我们证明妊娠期HBeAg阴性乙肝病毒携带者NK细胞分泌的IFN-γ可以显著抑制Th17细胞的极化，而HBeAg阳性妊娠期乙肝患者的NK细胞分泌IFN-γ水平降低。总之，本课题的研究将为妊娠期慢性乙型肝炎的防治提供实验基础和重要依据。