库普弗细胞条件培养基对原代培养肝细胞的细胞毒性

The cytotoxic effects of Kupffer cell-conditioned medium on primary cultured rat hepatocytes

目的探讨内毒素血症时库普弗细胞激活对肝细胞损伤的作用机制。方法用链霉蛋白酶和胶原酶原位灌流，Percoll密度梯度离心分离、纯化SD大鼠肝细胞和库普弗细胞，制备内毒素（LPS）刺激的库普弗细胞的条件培养基（KCCM）并作用于肝细胞，检测肝细胞培养基丙氨酸转氨酶（ALT）、天门冬氨酸转氨酶（AST）、乳酸脱氢酶（LDH）的含量，四甲基偶氮唑蓝（M1T）法检测KCCM对大鼠原代肝细胞活性的影响。结果KCCM作用于肝细胞2h后，肝细胞培养基中ALT、AST及LDH含量均明显升高（P〈0．05），在2～4h内随时间延长ALT、AST及LDH含量均逐渐增加，呈明显的时间．效应关系；MTT结果显示，KCCM作用后12h、24h时间点对肝细胞具有明显损伤作用（P〈0．05）。结论内毒素诱导库普弗细胞释放的可溶性因子作用一定时间后可直接对肝细胞造成损伤。

Objective To study the roles of Kupffer cells activated by lipopolysaccharides （LPS） on rat primary cultured hepatocytes in endotoxemia. Methods Hepatocytes and Kupffer cells were isolated from liver of Sprague-Dawley rats by in situ perfusion with pronase and collagenase and density gradient eentrifugation with Percoll, and then were cultured. The Kupffer cells were stimulated by LPS and kuffer cell-conditioned medium （KCCM） was collected. After hepatocytes exposed to KCCM in different time, transaminase （ALT and AST） and lactate dehydrogenate （LDH） were measured in the supematants. Meanwhile, MTT colori metric assay was used to detect the effects of KCCM on hepatocytes. Results The concentrations of ALT, AST and LDH were increased gradually. There was significant time-dependent relationship between KCCM and release of transaminase and LDH in the supernatants of rat primary cultured hepatocytes from 2 h to 24 h. The results of MTT revealed that hepatocytes were significantly injuried at 12 h and 24 h（ P 〈 0.05）. Conclusion The soluble factors released by Kupffer ceils, which were activated by LPS, could cause direct hepatocytes injury.