Abstract: Objective To investigate the effects of the immune response mediated by NOD1 receptor in liver sinusoidal cells ( LSECs ) of mice. Method Firstly, We used the method of the collagenase perfusion, percoll density gradient centrifugation and anti-LSEC MicroBeads separation and purification to obtain the high purity LSECs. The constitutive expression of NOD1 in LSEC was detected by real-time RT-PCR. Then the LSECs were stimulated with the diaminopimelic acid ( C12-ieDAP ) , the cells and cell supernatant were collected for 6 hours and 20 hours. Total RNA was isolated from LSECs for detecting the mRNA of NOD1, RIP2, IL-6, TNF-α, CXCL-1, CXCL-2, CXCL-9, CXCL-10, CCL-2 and CCL-5 by qRT-PCR. The cytokines ( IFN-α, IFN-β, IFN-γ, TNF-α and IL-6 ) in the cells supernatant were detected by quantitative ELISA. Result Our data showed that NOD1 mRNA was highly expressed at baseline in primary LSEC, and that the activation of LSEC by the NOD1 ligand C12-ieDAP could induce up-regulation of NOD1 mRNA and its connector RIP2 mRNA. The mRNA levels of cytokines IL-6 and chemokines CXCL-1, CXCL-2, CXCL-9, CCL-2 and CCL-5 were up-regulated. Stimulation of NOD1 ligand C12-ieDAP with the concentration of 10 μg / mL induced the production of IFN-γ and IL-6 effector molecules, but not other cytokines. Conclusion In conclusion, NOD1 was functionally expressed in LSEC, could be activated by its ligand, and mediated the activation of NF-κB signaling pathway to produce IL-6 and IFN-γ effector molecules as well as a series of cellular inflammatory cytokines and chemokines.

Key words: NOD1, liver sinusoidal endothelial cell, diaminopimelic acid, signaling pathway

摘要： 目的 本研究旨在调查小鼠原代肝窦内皮细胞( LSEC)中 NOD1 受体介导的免疫应答效应。 方法 用胶原酶 灌注与抗-LSEC 免疫磁珠分离方法分离 C57BL / 6 小鼠 LSEC。 应用 real-time RT-PCR 检测基础水平 LSEC 中 NOD1 的表达。 用 C12-ieDAP 配体刺激 LSEC, 6、20 h 收集细胞及细胞上清液,定量 RT-PCR 检测 NOD1、RIP2、IL-6、TNF-α、CXCL-1、CXCL-2、CXCL-9、CXCL-10、CCL-2、CCL-5 表达水平;ELISA 检测细胞上清液中细胞因子 IFNα / β / γ、TNFα 与 IL-6 蛋白产量。 结果 基础状态下 LSEC 中 NOD1 呈高度表达。 NOD1 配体 C12-ieDAP 激活 LSEC 诱导 NOD1 mRNA 高表达及其接头分子 RIP2 mRNA 高表达,细胞因子 IL-6 mRNA 与趋化因子 CXCL-1、CXCL-2、CXCL-9、CCL-2 和 CCl-5 等 mRNA 水平上调;NOD1 配体 C12-ieDAP 10 μg / mL 刺激诱导 IFN-γ 和 IL-6 效应分子产生。 结论 NOD1 在 LSEC 中功能性表达,能被其配体激活,并介导 NF-κB 信号途径活化产生 IL-6 和 IFN-γ 效应分子及一系列细胞 炎症因子与趋化因子。

关键词: NOD1, 肝窦内皮细胞, 二氨基庚二酸, 信号通路