MSCs对急性肝衰竭Kupffer细胞的调控及机制研究

Mesenchymal stem cells (MSCs) are not only characterized as multipotent stromal cells with the capacity for both self-renewal and differentiation into mesodermal cell lineages, but also have immunomodulation potential by modulating both innate and adaptive immune responses. The immunoregulatory functions of MSCs should be more important for the treatment of acute hepatic failure（AHF）. It has been well know that Kupffer cells, the liver macrophagocyte, is a key link of endotoxin-induced liver injuries and hepatic failure, so we presume that the therapy of MSCs on AHF is results in the modulation of immune disturbance by inhibiting the functions of Kupffer cells. To validate the hypothesis, cell activity, cytokine, such as transforming growth factor beta (TGF-β), prostaglandin E2 (PGE2), necrosis factor-alpha (TNF-α), interferon-gamma（IFN-γ）, interleukin（IL）, will be examined in co-cultivation of MSCs, Kupffer cells and hepatocytes stimulated by polyinosinic acid:polycytidilic acid (poly i:c) or lipopolysaccharide (LPS) for the evaluation of regulation MSCs on Kupffer cells in vitro. Then, the Kupffer cell functional activity inhibition or cell exhaustion mouse AHF model will be prepared. MSCs will be transplanted in mice that with and without Kupffer cells exhaustion or functions inhibition to observe the effect of Kupffer cells for the treatment of MSCs on AHF. Further, some key cytokines and receptors related LPS signal transduction system will be investigated by antagonist or enhancement correspondingly. Final, we hope to explain the cell and molecule mechanism of MSCs for AHF therapy through Kupffer cells modulation and provide an immunoregulatory evidence of MSCs in the treatment of AHF.

已证实MSCs具有免疫调节作用，这在治疗急性肝衰竭（AHF）中尤为重要。Kupffer细胞是肝巨噬细胞，司职天然免疫功能，是内毒素所致肝损伤进而肝衰竭的关键环节，MSCs治疗AHF应与Kupffer细胞有必然的联系。推测MSCs主要通过对Kupffer细胞功能的抑制作用，调节免疫失调，使AHF恢复。为验证此假说，拟首先进行在聚肌胞/内毒素脂多糖(LPS)刺激、损伤肝细胞条件下的MSCs和Kupffer细胞混合培养，观测细胞活性、PGE2、TNF-α等细胞因子变化，体外评价MSCs对Kupffer细胞的调节作用；在此基础上，制备抑制/耗竭Kupffer细胞的AHF小鼠模型，体内移植MSCs，观察Kupffer细胞对MSCs治疗AHF作用的影响；围绕LPS信号转导系统，挖掘关键的细胞因子和受体，利用对应的拮抗剂和增强剂进行干预，阐释MSCs通过调节Kupffer细胞治疗AHF的细胞与分子机制。

急性肝衰竭（AHF）病情凶险，预后极差，内科治疗尚无良策。间充质干细胞（MSCs)具有的自我更新、增殖、多向分化以及免疫调节能力和特性，使其逐步成为AHF治疗新的手段。已知，Kupffer细胞是肝内的天然免疫细胞，是内毒素所致肝损伤进而肝衰竭的关键环节，因此，MSCs治疗AHF应与Kupffer细胞有必然的联系。.为验证MSCs可能通过对Kupffer细胞功能的调节而治疗肝衰竭的假想，本项目：①改进和优化了分离培养MSCs、Kupffer细胞、肝细胞的方法，进行了多种组织来源MSCs的鉴定、诱导分化；②系列观察了LPS刺激下MSCs、Kupffer细胞、肝细胞活性、功能、细胞因子分泌变化及相互作用；③比较并制备了药物性急性肝衰竭模型的方法，用MSCs-肝细胞混合3D培养支架进行了肝衰竭动物的治疗实验；④采用药物干预方法建立了体内抑制Kupffer细胞活性的方法，观察了抑制Kupffer细胞对动物肝衰竭和移植MSCs肝内定植的影响；⑤在抑制Kupffer细胞条件下进行了动物肝衰竭的MSCs治疗实验，验证了MSCs与Kupffer细胞相互作用。.本课题的重要实验结果、发现及科学意义如下：.1、建立了稳定分离获取、培养肝细胞、kupffer细胞、MSCs的方法，实现了体内、外诱导MSCs向肝细胞的分化，制备出简便可靠的实验性急性肝衰竭的动物模型，获得了细胞分化与功能维持良好的MSCs-肝细胞3D共培养支架型肝组织，移植治疗急性肝衰竭效果显著，为有关肝脏疾病的基础与临床治疗研究提供了较好的、可供借鉴的方法和手段。.2、肝细胞、kupffer细胞（包括替代细胞RAW264.7细胞）、MSCs在内毒素刺激条件下，体外相互作用的系列实验显示，内毒素激活kupffer细胞，释放炎性细胞因子是造成肝细胞损伤的重要因素之一，MSCs对kupffer细胞的激活与炎性因子释放的具有调节作用，这为肝衰竭的发病机制和MSCs治疗提供了重要的实验依据。.3、建立了体内有效抑制kupffer细胞的实验方法，发现抑制急性肝衰竭kupffer细胞能显著减轻肝损伤，增加移植MSCs的肝内定植。佐证了kupffer细胞在肝衰竭发病机制中的作用与地位，同时提示了kupffer细胞的状态对MSCs移植的影响。发现抑制kupffer细胞条件下，MSCs治疗肝衰竭效果更好，证明MSCs的疗效与kupffer细胞密