肝硬化患者中性粒细胞功能受损与内质网应激的相关性研究

Patients with liver cirrhosis (LC) have impaired neutrophil function. Neutrophils in patients with LC are in a state of fully activation, however, they can not function properly (decreased chemotaxis, phagocytosis and bactericidal capacity) in condition of bacteria infection. The impaired liver function, increased serum levels of ammonia,endotoxin and cytokines have been implicated in the dysfunction of neutrophils in patients with LC. Recent studies have found that endoplasmic reticulum stress (ER stress) of T cells resulted in respiratory burst (RB), ER stress of neutrophils impaired their phagocytosis and bactericidal capacity, suggesting ER stress may be involved in impaired neutrophils function in patients with LC. Therefore we hypothesize that the ER stress of neutrophils are activated in patients with LC by endotoxin and/or cytokines, ER stress of neutrophils impaired their phagocytosis and bactericidal capacity. We plan to investigate the ER stress and chemotaxis, phagocytosis and bactericidal capacity of neutrophils in patients with LC. We also hope to explore the effects of ER stress on the functions of neutrophils in vitro, aimed at finding the association of impaired neutrophil function with ER stress in patients with LC.

已知肝硬化患者中性粒细胞功能下降，表现在中性粒细胞处于持续激活状态，但在发生细菌感染时其吞噬、趋化及呼吸爆发（RB）功能下降，原因尚不清楚，迄今的研究发现与肝硬化患者血内毒素、细胞因子及氨浓度增加等多种因素有关。近几年的研究发现T淋巴细胞内质网应激（ER stress）通过内质网线粒体偶联激活线粒体RB，中性粒细胞 ER stress损伤其吞噬功能。因此我们假设 ER stress是各种原因引起肝硬化患者中性粒细胞功能下降的共同病变基础。本研究拟：1、探讨不同程度肝硬化中性粒细胞功能及 ER stress相关基因及蛋白表达；2、探讨肝硬化患者血清、氨、内毒素及炎症细胞因子对正常人中性粒细胞 ER stress的诱导及功能损伤；3、探讨不同 ER stress诱导剂对正常人中性粒细胞的功能损伤。以明确肝硬化患者中性粒细胞功能受损与ER stress的关联。

肝硬化患者容易发生细菌感染，中性粒细胞免疫功能障碍在其中起关键作用。迄今对肝硬化患者中性粒细胞功能障碍原因尚不清楚。肝硬化患者体内普遍存在内质网应激（Endoplasmic reticulum stress, ER Stress），因此在本研究我们假设在肝硬化患者中，ER Stress 是多种因素损伤中性粒细胞功能的共同机制，探索肝硬化患者中性粒细胞功能损伤与ER Stress 之间的相关性。采用荧光定量PCR及蛋白免疫印迹法（Western-blot, WB）检测内质网应激相关基因及蛋白表达；采用琼脂糖凝胶平板法检测中性粒细胞的趋化功能；流式细胞术检测中性粒细胞的呼吸爆发功能。我们证实了不同程度肝硬化及慢加急性肝衰竭（ACLF）患者中性粒细胞存在ER Stress, 与肝脏损伤程度相关；肝硬化及ACLF患者炎症细胞因子及内毒素，血氨水平显著升高，中性粒细胞趋化、吞噬及呼吸爆发功能降低，中性粒细胞C-X-C chemokine receptor 1 (CXCR1)、CXCR2表达下降，细胞外诱捕网（NETs）增加，用ER Stress 抑制剂 4-苯基丁酸（4-PBA）减轻ER Stress后其趋化、吞噬及呼吸爆发功能及CXCR1、CXCR2表达得到部分恢复，NETs下降，提示中性粒细胞ER Stress可能是其趋化、吞噬及呼吸爆发功能降低的原因之一，中性粒细胞ER Stress可能通过下调CXCR1、CXCR2损伤其趋化功能。进一步用siRNA 干扰XBP1s通路后发现中性粒细胞CXCR1、CXCR2的表达得到部分恢复，提示中性粒细胞ER Stress可能通过XBP1s通路下调CXCR1、CXCR2的表达。