pHGF对TNF-α体外介导小鼠肝细胞凋亡的抑制作用

目的：探讨促肝细胞生长因子（pHGF）对肿瘤坏死因子－α（TNF－α）介导小鼠原代肝细胞凋亡和坏死的抑制作用。方法：采用原位灌洗法分离小鼠肝细胞，体外培养后分别加入TNF-α、GalN、TNF－α＋GalN＋pHGF5h、10h、20h和30h后,观察小鼠肝细胞的形态及生化改变，测定培养上清液ALT和AST。结果：TNF－α单独作用于培养肝细胞，无肝细胞凋亡和坏死，经GalN致敏后，TNF－α可诱导大量肝细胞凋亡（10h ),随后（20h后）出现肝细胞坏死，其程度随培养时间延长和药物剂量的增加而明显。单独使用GalN也可造成肝细胞凋亡和坏死，但程度较TNF－α＋GalN合用轻，加用pHGF后可使肝细胞凋亡坏死明显减少，其效应在一定药物剂量范围内成正比。结论：pHGF对TNF－α诱导肝细胞凋亡和坏死具有抑制作用。

pHGF inhibits hepatocyte apoptosis induced by TNF-α in mice in vitro

Objective:To investigate the effects of inhibition of promotinghepatocyte growth factor(pHGF) on primary hepatocyte apoptosis and necrosis induced by tumor necrosis factor-α(TNF-α) in vitro.Methods:The hepatocytes were isolated in the way of liver perfusion in situ with 0.05% collagenase type Ⅳ and cultured with TNF-α、D-galactosamine(GalN)、TNF-α+GalN or TNF-α+GalN+pHGF respectively for 5 h、10 h、20 h and 30 h.Hematoxylin and esoin(HE)、Hoechst 33 258 staining were used to observe the morphological changes of hepatocyte.DNA fragment was analyzed by the agarose gel electrophoresis to determine the hepatocyte apoptosis.The ALT(alanine aminotransferase) and AST(aspartate aminotransferase) activities of cultured supernatant were measured for assessing necrosis.Results:Hepatocytes incubated with TNF-α+GalN showed typical apoptosis specific morphological feature,the number of hepatocyte apoptosis increased in parallel with the dose of TNF-α and incubation time.The hepatocyte apoptosis was further confirmed by DNA ladder.In the late time(after 20 h),hepatocyte necrosis took place with the increase of ALT and AST.In addition,hepatocyts incubated with GalN also caused small ammounts of hepatocyte apoptosis and necrosis.The hepatocyte apoptosis and necrosis in TNF-α+GalN+pHGF group were decreased significantly.Conclusions:pHGF can inhibit primary hepatocyte apoptosis and necrosis induced by TNF-α in vitro.