Qiu De Kai, Hua Jing, Li Ji Qiang, Li Eng Ling
Institute of Digestive Disease, Renji Hospital, Shanghai Second Medical University, Shanghai, China.
Chin J Dig Dis. 2005;6(3):137-41. doi: 10.1111/j.1443-9573.2005.00208.x.
To investigate the expression of CD14 on Kupffer cells during the course of carbon tetrachloride (CCl(4))-mediated liver injury and its role in the activation of Kupffer cells.
Rats were administered CCl(4) twice weekly for up to 8 weeks. Kupffer cells were isolated from normal and CCl(4)-treated rats by the combined 'collagenase-pronase' perfusion method, discontinuous density gradient centrifugation. On the day after isolation, the cells were incubated with RPMI-1640 containing varying doses of lipopolysaccharide (LPS) for 6 h. Supernatants were then collected for measuring the concentration of tumor necrosis factor-alpha (TNF-alpha) by enzyme-linked immunosorbent assay (ELISA). The expression of CD14 mRNA on Kupffer cells were determined by RT-PCR. The plasma concentrations of endotoxin were determined by chromogenic substrate Limulus amebocyte lysate assay.
Basic TNF-alpha production of Kupffer cells isolated from CCl(4)-treated rats at 4 and 6 weeks was significantly higher than that of normal (P < 0.05). Following LPS stimulation the production of TNF-alpha was markedly increased in Kupffer cells from the 2-, 4- and 6-week treatment groups (P < 0.05). Moreover, LPS-induced TNF-alpha production was dose-dependent. 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 6 weeks. Gene expression was decreased in Kupffer cells after 8 weeks of CCl(4) treatment. CCl(4) administration elicited extensive changes in liver morphology, including steatosis, inflammation and necrosis. The plasma concentrations of endotoxin of CCl(4)-treated rats were increased during the time of liver injury.
Up-regulation of CD14 expression in Kupffer cells during CCl(4)-mediated chronic liver injury indicates cell activation and that they are more sensitive to LPS stimulation. Kupffer cells are critical effector cells in the early stage of liver injury.
研究四氯化碳（CCl₄）介导的肝损伤过程中枯否细胞上CD14的表达及其在枯否细胞激活中的作用。
大鼠每周两次给予CCl₄，持续8周。采用“胶原酶-链霉蛋白酶”联合灌注法、不连续密度梯度离心法从正常和CCl₄处理的大鼠中分离枯否细胞。分离后次日，将细胞与含不同剂量脂多糖（LPS）的RPMI-1640孵育6小时。然后收集上清液，用酶联免疫吸附测定（ELISA）法测定肿瘤坏死因子-α（TNF-α）浓度。通过逆转录聚合酶链反应（RT-PCR）测定枯否细胞上CD14 mRNA的表达。用显色底物鲎试剂法测定血浆内毒素浓度。
在第4周和第6周，从CCl₄处理的大鼠中分离的枯否细胞基础TNF-α产生量显著高于正常组（P<0.05）。LPS刺激后，第2周、第4周和第6周处理组的枯否细胞中TNF-α产生量明显增加（P<0.05）。此外，LPS诱导的TNF-α产生呈剂量依赖性。给予CCl₄ 2周后，从CCl₄处理的大鼠中分离的枯否细胞上CD14 mRNA表达升高，最大升高出现在第6周。CCl₄处理8周后枯否细胞中基因表达下降。给予CCl₄引起肝脏形态广泛改变，包括脂肪变性、炎症和坏死。CCl₄处理的大鼠在肝损伤期间血浆内毒素浓度升高。
CCl₄介导的慢性肝损伤过程中枯否细胞CD14表达上调表明细胞被激活，且它们对LPS刺激更敏感。枯否细胞是肝损伤早期的关键效应细胞。
