内毒素／肿瘤坏死因子所致肝细胞损害机制的体外研究

An in vitro study of mechanisms on lipopolysaccharide/tumor necrosis factor-induced hepatocyte in-jury in rats

为了解内毒素（ＬＰＳ）／肿瘤坏死因子（ＴＮＦ）所致肝细胞损害的机制，以乳酸脱氢酶（ＬＤＨ）及噻唑蓝染色（ＭＴＴ）为细胞毒性指标，利用Ｗｉｓｔａｒ大鼠肝细胞进行了ＬＰＳ及其介质ＴＮＦ等的肝细胞损害机制研究。结果发现：ＬＰＳ、ＴＮＦ－α、ＩＬ－１、ＩＬ－６等对肝细胞ＬＤＨ漏出及ＭＴＴ无显著影响（Ｐ＞０．０５），仅在ＬＰＳ、ＬＰＳ／ＴＮＦ－α加入肝细胞－Ｋｕｐｆｆｅｒ细胞混合培养组后有所变化；经ＧａｌＮ／ＬＰＳ体内作用后分离的Ｋｕｐｆｆｅｒ细胞与正常肝细胞混合培养，加入ＬＰＳ、ＴＮＦ及ＬＰＳ／ＴＮＦ－α均可致ＬＤＨ漏出显著增高（Ｐ＜０．０ｌ），多粘菌素Ｂ及抗－ＴＮＦ能分别完全和部分阻断此效应；经ＬＰＳ或ＴＮＦ－α体内作用后抽取的大鼠血清，加入培养肝细胞可致ＬＤＨ漏出显著增高（Ｐ＜０．０１）与ＭＴＴ显著降低（Ｐ＜０．０５），经５６℃灭活后此细胞毒性作用完全消失。上述结果提示，ＬＰＳ及其介质ＴＮＦ无肝细胞直接毒性作用，而经其活化的Ｋｕｐｆｆｅｒ细胞可能引起一系列瀑布效应，导致肝细胞损害。

This experiment was carried out to explore the mechanisms of lipopolysaccharide(LPS)or/and tumor necrosis factor(TNF)-induced hepatocyte injury in cultured liver cells in Wis-tar rats.The cytotoxicity was determined by lactate dehydrogenase(LDH)release and MTT,Nosignificant changes could be found in LDH release and MTT of hepatocytes after the addition of LPS,TNF-αinterlukin(IL)-1and IL-6(P>0.05),but there were somewhat changes in coculture of hepatocyte-Kupffer cell after the addition of LPS and LPS/TNF-α(P<0.l>0.05).When hepa-tocytes were cocuItured with the Kupffer cells separated from the rats pretreated with D-galac-tosamine(GalN)nd LPS,increased LDH release and decreased MTT occurred after the additions of LPS,TNF-αand LPS/TNF-α(P<0.01).The changes could be completely and partially blocked by polymyxin B and anti-TNF-αin turn。 The serum from LPS or TNF-α treated rats,prepared after intravenous administrations of LPS and TNF-αrespectively,caused a significant increase of LDH re-lease and decrease of MTT of hepatocytes(P<0.0l).The cytotoxicity disappeared when the serum from LPS or TNF-αtreated rats was pretreated by heating at 56℃ for 30 min.。The above mentioned results mentioned above suggest that LPS and cytokines such as TNF have no direct cytotoxicity on hepatocytes,but the Kupffer cells activated by LPS or TNF-α may induce hepatocyte injury through aseries of cascade effects in vivo.