Ces1f的枯否细胞极化效应在急性肝衰竭肝组织炎症损伤中的作用研究

Acute liver failure (ALF) is a clinical severe disease characterized by rapid liver inflammatory injury and high mortality rates; however, the related mechanism is not clear. Kupffer cells (KCs) are the main innate immune cells in the liver, and its inflammatory polarization phenotype (M1) plays a povital role in the pathogenesis of ALF. In the previous study, we found significant upregulation of Ces1f expression in LPS-stimulated primary KCs and in the liver tissues of ALF. From here we reason that Ces1f may mediate hepatic inflammatory injury through promoting M1 switch of KCs in ALF. To verify the above inference, in this study, Ces1f KO mice will be used to investigate the relationship between Ces1f and hepatic damage of ALF and the effect of Ces1f on KC polarization, and further to probe the role of KC polarization of Ces1f in the pathogenesis of ALF via macrophage removal and adoptive transfer. At the same time, primary KCs and macrophage cell lines will used to study the effects of Ces1f on cellular polarization phenotype, inflammatory release, metabolic cascades and protein expression spectrum, and the effects on biological activity of common-cultured hepatic cells in LPS-stimulated macrophages. Lastly, clinical hepatic specimens from ALF patients will be used to assay Ces1f expression and polarization phenotype of KCs and protein expression profiles of liver. Such a study provides an experimental basis for the probe of injury mechanism of Ces1f and the clinical regulation of immune inflammation in ALF.

急性肝衰竭（ALF）是临床急危重症，病死率高，发病机制不明。KC是肝主要的固有免疫细胞，其向炎症极化（M1）转变在ALF发生中起关键作用。我们前期研究发现，LPS刺激KC内Ces1f表达上调，且ALF肝内Ces1f表达也上调。由此推测，Ces1f可能通过KC的M1转变介导ALF肝炎症损伤。为验证以上推论，本项目将利用Ces1f敲除小鼠，通过巨噬细胞去除和过继转移手段，观察Ces1f与ALF肝损伤之间的关系及对KC极化的影响，分析Ces1f的KC极化效应在ALF发生中的作用；同时，利用原代KC和巨噬细胞系，观察Ces1f对LPS刺激巨噬细胞极化表型、炎症释放、代谢级联和蛋白质表达谱的影响以及对共培养肝细胞生物学活性的影响；最后，观察ALF病人肝和KC Ces1f表达、KC极化和肝蛋白表达谱情况。项目研究完成可为Ces1f在ALF炎症损伤机制研究和临床免疫炎症调控提供实验基础。

急性肝衰竭（ALF）是肝组织炎性损伤性疾病。枯否细胞（KC）在肝脏固有免疫反应中起重要作用，其极化表型与功能异常，可引发抗炎性M2表型向炎症性M1表型转变，造成肝组织免疫炎症性损伤。我们前期芯片研究证实，酯代谢酶分子Ces1f在炎性刺激KC内存在显著差异性表达。在本项目中，我们研究了Ces1f的KC炎性极化效应及其对ALF的影响。我们首先构建了Ces1f-siRNA，并通过基因重组和兔免疫实验等方法制备了Ces1f多克隆抗体。为达到定向敲减活体动物枯否细胞目的基因表达的目的，我们采用了葡聚糖包裹Ces1f-siRNA分子（即GeRPs）的方法，采用鼠尾静脉注射，实现了Ces1f-siRNA活体动物KC靶向转移和目的基因定向敲减；在对ALF小鼠的研究中，我们发现ALF肝内Ces1f的水平伴随着UII的上调而显著下降。当采用urantide阻断UII/UT信号传导后，ALF肝内Ces1f的下调被逆转，同时肝组织炎性损伤明显减轻；当采用GeRPs方法对肝枯否细胞目的基因进行定向敲减后，我们发现，小鼠肝内Ces1f表达下调，并与ALF肝组织Ces1f受抑相叠加，造成肝内Ces1f水平显著降低，动物死亡率增加，肝细胞凋亡和肝损伤显著加重，促炎细胞因子如TNF-α和IL-6表达和分泌水平显著升高，而抑炎细胞因子如IL-10分泌明显降低；另外，小鼠活体肝枯否细胞Ces1f基因定向敲减，也造成了ALF枯否细胞极化表型与功能失调，促炎性M1表型细胞显著增多，其极化标记物如CD86和iNOS表达显著增高；而抑炎性M2表型细胞明显减少，其极化标记物如CD163和Arg-1表达也显著降低，即发生M2向M1极化表型的转变。伴随这种极化表型转变，ALF肝组织氧化应激反应和炎性损伤程度也显著加重。这表明，Ces1f可能有助于维持肝内枯否细胞M2极化表型的稳定，减少或阻止M2向M1转变，进而防止ALF的发生。该项成果的取得，有望更好地阐明ALF发生的免疫损伤机制。通过对物质代谢途径的调控，将来有可能实现枯否细胞极化稳定，并达到肝组织损伤修复的目的。因此，项目具有较好的理论和临床实用价值。