库普弗细胞内毒素受体CD14在大鼠肝损伤中的变化

The expression of CD14 on Kupffer cells during the course of carbon tetrachloride-induced liver injury

目的动态观察四氯化碳(CCl4)诱导大鼠肝损伤过程中库普弗细胞膜上脂多糖(LPS)受体CD14表达变化及其在细胞因子分泌中的作用。方法以CCl4皮下注射诱导大鼠肝损伤。采用联合酶灌注消化、不连续密度梯度离心法分离不同时期大鼠肝脏库普弗细胞。将库普弗细胞与不同浓度内毒素孵育6h,收集培养上清并抽提细胞RNA。ELISA检测细胞培养上清中肿瘤坏死因子(TNF)α水平。RTPCR法检测库普弗细胞CD14mRNA表达。偶氮基质显色法测定大鼠血浆内毒素水平。结果经CCl4处理4周和6周,大鼠库普弗细胞在体外培养时TNFα基础分泌量明显高于正常大鼠(P<0.05)。在LPS刺激下,CCl4处理2、4和6周库普弗细胞的TNFα分泌水平明显增加(P<0.05),呈浓度依赖方式。CCl4组大鼠库普弗细胞CD14mRNA表达从2周开始增加,6周时最明显,8周时恢复至正常水平。大鼠外周血内毒素浓度在肝损伤过程中升高。结论在CCl4诱导的大鼠慢性肝损伤早期过程中,库普弗细胞内毒素受体CD14表达上调,对内毒素敏感性增加。库普弗细胞是肝脏早期炎症反应中的一个重要效应细胞。

Objective To investigate the expression of CD14, the receptor of lipopolysaccharide (LPS) on Kupffer cell membrane during the course of CCl4-induced liver injury and its role in activation of Kupffer cells. Methods The experimental rats were hypodermically administered CCl4 twice weekly for up to 8 weeks. Kupffer cells were isolated from the liver of normal and CCl4-treated rats by the combined ‘collagenase-pronase’ perfusion method and discontinuous density gradient centrifugation. On the following day after isolation, the cells were incubated with RPMI-1640 containing various doses of LPS for 6 h. The tumor necrosis factor (TNF-α) in the supernatants was measured by ELISA. The expression of CD14 mRNA on Kupffer cells was determined by RT-PCR. The plasma levels of endotoxin were determined by chromogenic substrate limulus amebocyte lysate assay. Results Baseline TNF-α production of Kupffer cells isolated from CCl4-treated rats in 4 and 6 weeks was significantly higher than that of controls (P<0.05). With LPS stimulation, the production of TNF-α was dose-dependently increased in Kupffer cells from 2, 4 and 6 weeks in the CCl4-treated group (P<0.05). CD14 mRNA expression on Kupffer cells isolated from CCl4-treated rats was elevated following 2 weeks of CCl4 administration and the maximum elevation occurred at the 6th week time point, and then decreased to normal at the 8th week. The levels of plasma endotoxin of CCl4-treated rats were increased during the course of liver injury. Conclusions Up-regulation of CD14 expression in Kupffer cells during CCl4-induced chronic liver injury may indicate the cell activation and more sensitive to LPS stimulation. Kupffer cells are critical effector cells in the early stage of liver injury.