新生鼠肝脏巨噬细胞－自然杀伤细胞双向调控功能异常致胆道闭锁的机制研究

Both macrophages and natural killer cells (NK cell) in liver play an important role in the pathogenesis of biliary atresia induced by rotavirus(RRV) in mice,however,the dysfunctional crosstalk between neonatal macrophages and NK cells in neonatl period in the development of biliary atresia is still unknown and has never been reported.In this study,macrophages and NK cells collected from mice liver in different age will be cocultured in vitro in the presence of RRV and RRV-infected biliary epithelial cells. With the purpose to illustrate the key factor that neonatal macrophages modulate NK cells, molecular interactions that regulate the macrophage-dependent activation of NK cells in newborn mice will be mainly focused on cytokines, receptor-ligand interactions and immune synapses, by comparatively analyzing the ability of macrophages that have been primed with RRV or RRV-infected biliary epithelial cells at different ages to release chemokines, cytokines and express ligands for corresponding activating receptors of NK cells.In the following, difference cytotoxicity of NK cells at different ages against RRV-infected macrophages will be comparatively analyzed. Triggered by IFN-γ,IL-4,and IL-13 released from NK cells, the differential activation of macrophages at different ages will be comparatively analyzed to illustrate the key point that neonatal NK cells modulate NK cells cell-dependent activation of macrophages.Meanwhile,the relationship of dysfunctional crosstalk in macrophages and NK cells will be explored in vivo in newborn mice.According to the research, It is expected for the first time to elucidate the definite pathogenesis of biliary atresia only occured in neonates , and contribute to the theory of preventing biliary atresia occurrence.

肝脏巨噬细胞（MΦ）和自然杀伤细胞（NK）在轮状病毒引起的鼠胆道闭锁中起重要作用，但新生鼠MΦ和NK细胞的功能均不成熟，二者相互调控异常在轮状病毒引起鼠胆道闭锁中的作用尚未见报道。本课题拟观察不同年龄鼠肝脏MΦ在吞噬轮状病毒及轮状病毒感染的胆管上皮细胞后释放趋化因子、细胞因子及表达NK细胞活化受体之配体的能力，从细胞因子、活化受体配体表达及免疫突触形成三个方面明确新生鼠肝脏MΦ对肝脏NK细胞调控作用的关键点。通过比较不同年龄鼠肝脏MΦ在肝脏NK细胞分泌的IFN-γ、IL-4及IL-13作用下的活化程度，并观察不同年龄鼠肝脏NK细胞对吞噬轮状病毒的肝脏MΦ的杀伤作用，从而明确新生鼠肝脏NK细胞对肝脏MΦ免疫功能的调控特点。同时在体实验中探讨新生鼠肝脏MΦ和NK细胞相互调控功能异常与胆道闭锁形成间的关系。通过以上研究有望首次弄清胆道闭锁特发于新生儿期的原因，为预防胆道闭锁的发生奠定理论基础。

研究背景：胆道闭锁是一种特发于新生儿期的肝内外胆管进行性、闭塞性病变，该病进展迅速，很快发生纤维化，如不进行有效治疗，患儿将在1岁左右死亡。胆道闭锁的病因尚不明确。肝脏巨噬细胞（Mφ）和自然杀伤细胞（NK）在轮状病毒引起的鼠胆道闭锁中起重要作用，但新生鼠Mφ和NK细胞的功能均不成熟，二者相互调控异常在轮状病毒引起鼠胆道闭锁中的作用尚未见报道。研究内容：本课题拟观察不同年龄鼠肝脏Mφ在吞噬轮状病毒及轮状病毒感染的胆管上皮细胞后释放趋化因子、细胞因子及表达NK细胞活化受体之配体的能力，明确新生鼠肝脏Mφ对肝脏NK细胞调控作用的关键点；通过比较不同年龄鼠肝脏Mφ在肝脏NK细胞分泌的IFN-γ、IL-4、IL-13作用下的活化程度，并观察不同年龄鼠肝脏NK细胞对吞噬轮状病毒的肝脏Mφ的杀伤作用，明确新生鼠肝脏NK细胞对肝脏Mφ免疫功能的调控特点；同时在体实验中探讨新生鼠肝脏Mφ和NK细胞相互调控功能异常与胆道闭锁形成间的关系。结果：完成了肝脏巨噬细胞、NK细胞以及肝外胆管上皮细胞3种原代细胞的分离、原代培养及transwell培养，方法稳定；新生小鼠肝脏MΦ在RRV刺激下表达IL-12、IL-23、TNF-α及CD40的水平明显低于成年小鼠，而Rae-1、IL-10的表达高于成年小鼠；共培养IL-12和IL-10中和实验进一步发现IL-12及IL-10对成年小鼠NK细胞表达TNF-α的正向及负向作用均大于新生小鼠；共培养后Rae-1和CD40阻断实验表明肝脏MΦ-NK之间的Rae-1-NKG2D、CD40-CD154信号有助于新生小鼠NK细胞IFN-γ及TNF-α的表达；通过过继转移细胞实验，发现将成年小鼠的肝脏NK细胞转移至B6新生小鼠，注射轮状病毒14天后，观察各组小鼠的胆管损伤分级，胆管损伤1级以上的小鼠数量明显下降。结论：IL-12并非决定NK细胞胞内IFN-γ表达水平的关键因素，而新生小鼠IL-10对NK细胞IFN-γ的调控不足可能是发生BA的重要原因；Rae-1/NKG2D的信号通路在新生小鼠NK细胞中的过度活化，可能是导致BA的一个重要信号通路；新生鼠肝脏MΦ或NK细胞功能不足是胆道闭锁发生于新生儿期的原因。