MSCs抑制循环单核细胞活化挽救急性肝衰竭猴模型的机制研究

Acute liver failure (ALF) can occur as a result of various etiologies with sophisticated pathogenesis that has not been fully elucidated. It is well known that the disorder of the innate immune cells including Kupffer cells (KCs) plays a dominant role in the initiation, progression and prognosis of ALF. We previously established a drug-induced non-human primate ALF model and unexpectedly observed that during the first 24 h, KCs were only slightly activated. However, serum IL-6, the crucial pro-inflammatory cytokine and acute phase reactive protein that mainly produced by macrophages, was robustly increased and was much earlier than dozens of other cytokines. Most intriguing, IL-6 was rapidly decreased accompanied with the extensive KCs infiltration and activation, suggesting that IL-6 is not uniquely produced by KCs. When the monkeys were treated with peripheral infusion of human umbilical cord mesenchymal stem cells (hUC-MSCs), their liver function, hepatic histopathology and survival were significantly improved and the activation of KCs, serum levels of IL-6 as well as other inflammatory cytokines were tremendously reduced. Circulating monocytes can migrate into liver and different into KCs when they are chemo-attracted by local damage and are regarded as the major source of KCs. It was reported that circulating monocytes could not be activated until they different into KCs, however, our findings demonstrated that circulating monocytes are likely extensively activated before they migrate into liver, and act as IL-6 producers and play a critical role in the exacerbation of systemic inflammation. Furthermore, we speculate that circulating monocytes are the target cells of hUC-MSCs. In this study, we aim to elucidate the activation of circulating monocytes during the full course of ALF and investigate whether and how hUC-MSCs inhibit the function of the circulating monocytes.

急性肝衰竭（ALF）发病机制复杂，以Kupffer细胞（KCs）为主的先天性免疫系统紊乱至为关键。然而在我们建立的药物性猴ALF模型中，病程启动24h内KCs活化轻微，而血清中重要的促炎急性期反应蛋白IL-6骤升近百倍，远早于其他数十种因子；KCs大量浸润后IL-6却急剧下降，提示IL-6并非单纯来源于KCs。经外周血输入人脐带间充质干细胞（hUC-MSCs）能显著改善ALF猴肝功能和组织学，强烈抑制KCs活化及IL-6等多种炎症介质。KCs主要来自循环单核细胞，后者迁移并分化为KCs后才被激活，但我们推测其在入肝前已活化，是IL-6的主要来源和系统性炎症的重要参与者，也极可能是hUC-MSCs调控的靶细胞。本研究将探讨ALF时循环单核细胞活化规律，证实hUC-MSCs抑制循环单核细胞活化的作用及其机制，以期阐明ALF发生发展及转归规律，为将hUC-MSCs用于临床ALF治疗奠定基础。

我们采用大型非人灵长类动物模型，通过腹腔注射鹅膏蕈碱和低剂量LPS，建立了稳定的急性肝衰竭（ALF）模型。采用定期活检、液相芯片、免疫组化、流式细胞等技术，系统动态地揭示了ALF启动、进展及最后致死过程中的炎症细胞和炎症因子的动态变化，首次揭示了循环单核细胞在该过程中的关键核心作用，也为治疗由中毒等原因引起的ALF提供了靶点。本模型也为探讨ALF机制、开发治疗手段等提供了很好的模型。通过外周血输入hUC-MSCs，能显著改善动物肝组织学结构、肝功能指标以及系统性炎症反应。在排除了hUC-MSCs分化为肝细胞、促进肝再生、免疫调节等可能机制后，我们发现hUC-MSCs可显著抑制单核细胞的活化，抑制其分泌IL-6，从而在早期掐断细胞因子风暴的级联放大，有效避免肝损伤导致的SIRS发生发展，达到治疗ALF的作用。尽管我们的研究提示hUC-MSCs需要尽早输入，但我们还发现即使错过了最佳治疗时间窗，给与大剂量hUC-MSCs仍然有较好疗效。通过模拟体内炎性环境，我们发现hUC-MSCs在IL-6刺激下大量分泌外泌体，而这些外泌体可抑制单核-巨噬细胞活化。进而我们鉴定出miR-455-3p 这一分子，分别在内毒素模型和化学性肝损伤模型中证实其具有抑制单核细胞活化、改善局部损伤和系统性炎症反应的作用。本项目已发表SCI论文3篇，申报发明专利一项。同时，我们认为hUC-MSCs具有较好的安全性，其有效性也得到了验证，具备了向临床转化的条件。课题组在此基础上，联合我院急诊科，与四川省干细胞库/四川新生命干细胞有限公司联合开展了“hUC-MSCs治疗毒蕈中毒性肝损伤临床前研究”项目。我们纳入60只恒河猴，建模后24h给与单次hUC-MSCs外周血输入。本实验尽可能模拟了临床实际情况，在疾病已充分发展后再实施治疗。目前本合作项目已完全部实验，其结果显示了hUC-MSCs在治疗中毒性肝损伤（肝衰竭）中的良好前景。本项目后续研究还得到了四川省科技厅应用基础研究重大前沿项目和四川省卫健委重点项目支持，已通过我院干细胞临床项目伦理审查，在完成备案所需各项材料和程序后将开展临床试验。