摘要
目的探讨内毒素、肿瘤坏死因子（ＴＮＦ）在诱导枯否细胞及肝细胞凋亡过程中的作用及其与肝脏损伤的关系。方法采用细胞死亡ＥＬＩＳＡ法分别检测枯否细胞，以及和肝脏细胞联合培养时，对不同浓度的内毒素，在不同时相点两种细胞的凋零密度，同时观察培养上清中丙氨酸转氨酶（ＡＬＴ），乳酸脱氢酶（ＬＤＨ）含量变化。结果枯否细胞的凋亡数量随着培养时间的延长（３～２４小时）和内毒素浓度的增加（０～１０μｇ／ｍｌ）明显增加。而枯否细胞与肝细胞联合培养时，在内毒素浓度大于１μｇ／ｍｌ时，细胞凋亡数显著上升。ＴＮＦ单克隆抗体可阻断内毒素诱导的枯否细胞及肝细胞凋亡。ＬＤＨ及ＡＬＴ含量在内毒素浓度高于１μｇ／ｍｌ，且培养６小时后，有明显升高，晚于凋亡出现。结论ＴＮＦ介导了内毒素所致的枯否细胞及肝细胞的凋零。而且细胞凋亡早于细胞损伤的出现。大量凋零细胞的出现，使枯否细胞对内毒素的灭活作用降低进而可能导致肝细胞的凋零及损伤。
Objective To study the role of tumor necrosis factor (TNFα) in the mechanisms of liver injury and the relationship between LPS induced apoptosis in hepatocyte (HC), kupffer cell (KC), and hepatic damage. Methods In vitro, kupffer cells (1×10/ml) were stimulated with different concentrations of endotoxin (1～10 μg/ml) for 1 24hr; the kupffer cells, which had been cultured with endotoxin for 24hr, were isolated to coculture with hepatocytes (1∶1) for 1～24hr. Two kinds of cells of apoptosis and the block effect of TNFα antibody were detected by cell death ELISA, and the supernatant was performed to determine the levels of alanine aminotransferase (ALT), lactate dehydrogenase (LDH) at different time points. Results Following stimulation with LPS, the number of positive apoptotic KC increased in a concentration and time (3h～24h) dependent manner. In contrast, coincubation of HC with LPS stimulated KC resulted in a marked increase in positive apoptotic hepatocyte when LPS concentration was greater than 1 μg/ml. TNFα antibody blocked apoptosis in both KC and HC. Only when the LPS concentration was higher than 1 μg/ml and cultured for 6hr, the release of liver enzymes (ALT, LDH), responsible for hepacyte damage, rose significantly, but the process was behind of apoptosis, and TNFα antibody couldn′t block it. Conclusion TNFα mediates LPS induced apoptosis in KC and HC, and the apoptosis precedes cellular damage. Massive apoptosis of KC may lead to the decrease of clearance of LPS, thereby exacerbating septic shock, hepatocyte damage and apoptosis.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1998年第6期423-425,共3页
National Medical Journal of China
基金
国家自然科学基金
