α-GalCer诱导急性肝损伤时肝内Kupffer细胞表型和功能的变化

Phenotypic and Functional Changes of Kupffer Cells in a-GalCer Induced Acute Liver Injury

背景:Kupffer细胞是肝内重要的免疫细胞,在天然免疫和适应性免疫中起重要作用。目的:研究α-GalCer诱导的急性肝损伤中肝内Kupffer细胞表型和功能变化。方法:将30只小鼠分为模型组和对照组,腹腔注射α-GalCer诱导小鼠急性肝损伤。行肝组织病理学检查,免疫组化法检测肝内F4/80阳性的Kupffer细胞的分布。胶原酶原位灌注、不连续密度梯度离心和选择性贴壁法分离正常和急性肝损伤小鼠的Kupffer细胞。real time-PCR法检测肝组织和Kupffer细胞肿瘤坏死因子(TNF)-α、于扰素γ(IFN-γ)、白细胞介素(IL)-10和Toll样受体(TLR)4mRNA表达。采用全自动生化仪测定血清ALT和AST水平。结果:α-GalCer腹腔注射引起小鼠局灶性肝细胞坏死、炎性细胞聚集,血清ALT、AST明显升高(P<0.05)。免疫组化法发现肝内F4/80阳性细胞明显增加、体积增大,并在肝组织炎症坏死处呈灶性聚集。胶原酶原位灌注分离的Kupffer细胞数量明显增加(P<0.01),肝组织和Kupffer细胞TNF-α、IFN-γ和IL-10 mRNA表达明显增加(P<0.05),而Kupffer细胞中TLR4 mRNA表达显著下降(P<0.05)。结论:在急性肝损伤中Kupffer细胞的抗炎因子IL-10表达增加和TLR4途径下调可能是Kupffer细胞维持机体免疫耐受、避免过度炎症的重要机制。

Kupffer ceils are important immune ceils in liver, which play important roles in innate immunity and adaptive immunity. Aims： To investigate the phenotypic and functional changes of Kupffer cells in acute liver injury induced by α-GalCer. Methods： Thirty mice were divided into model group and control group. Acute liver injury was induced by intraperitoneal injection of α-GalCer in mice. Histopathologic examination of liver tissue was performed, and F4/80 positive Kupffer cells were detected by immunohistochemistry. Kupffer cells were isolated by in situ perfusion of collagenase, discontinuous density gradient centrifugation and selective adherence method from normal and acute liver injury mice. Tumor necrosis factor ct （TNF-α） , interferon y （IFN-y） , interleukin 10 （IL-10） and Toll-like receptor 4 （TLR4） mRNA expressions in liver tissue and Kupffer cells were detected by real time-PCR assay. Serum ALT and AST levels were measured by automatic biochemistry analyzer. Results： Focal necrosis of hepatocytes and aggregation of inflammatory ceils were detected after administration of cL-GalCer, accompanied by rise of serum ALT and AST levels （ P 〈 0.05）. F4/80 positive Kupffer cells increased markedly, enlarged in size, and gathered focally in inflammatory necrosis sites. Number of Kupffer cells isolated increased significantly （P 〈 0. Ol ）. The mRNA expressions of TNF-ct, IFN-y and IL-10 in isolated liver tissue and Kupffer cells were markedly increased （ P 〈 0.05 ）, while mRNA expression of TLR4 in Kupffer cells was markedly decreased （P 〈 0.05 ）. Conclusions： Production of IL-10 is increased and TLR4 signal is down-regulated in Kupffer ceils in acute liver injury. This might be the mechanism for maintaining immune tolerance and avoiding excessive inflammatory response in acute liver injury.