摘要
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 CCl4-induced liver injury and its role in activation of Kupffer cells. Methods The experimental rats were hypodermically administered CCl4 twice weekly for up to 8 weeks. Kupffer cells were isolated from the liver of normal and CCl4-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 CCl4-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 CCl4-treated group (P<0.05). CD14 mRNA expression on Kupffer cells isolated from CCl4-treated rats was elevated following 2 weeks of CCl4 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 CCl4-treated rats were increased during the course of liver injury. Conclusions Up-regulation of CD14 expression in Kupffer cells during CCl4-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.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2005年第6期340-343,共4页
Chinese Journal of Digestion