肝衰竭患者血浆对HepG2细胞增殖的抑制及表皮生长因子的调控作用

目的:探讨肝衰竭患者血浆(liver failure plasma,LFP)抑制HepG2细胞生长、增殖的机制及表皮生长因子(epidermal growth factor,EGF)对其抑制作用的调控作用.方法:通过用50%LFP与HepG2细胞共同培养(肝衰竭组),以相同浓度正常人血浆(正常对照组)作对照.采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)、Hoechst法分别观察不同时间点细胞增殖率及凋亡率,并用Western blotting法检测细胞周期蛋白Cyclin D1、细胞周期蛋白依赖激酶4(cyclin-dependent kinase4,CDK4)表达.进一步观察不同浓度EGF对LFP培养的HepG2细胞生长及增殖的刺激作用.结果:50%LFP对HepG2细胞生长及增殖有明显的抑制作用,且呈时间依赖性.EGF对正常对照组HepG2细胞的生长及增殖有明显促进作用,但仅大剂量EGF对肝衰竭组HepG2细胞生长和增殖有一过性刺激作用.与EGF刺激正常对照组比较,肝衰竭组各时间点细胞增殖仍受到明显抑制.LFP与HepG2细胞培养后,细胞凋亡率无明显增加(P>0.05).随着作用时间的延长,LFP抑制HepG2细胞内Cyclin D1、CDK4的表达.结论:LFP对HepG2细胞生长及增殖具有较强的抑制作用,但并不明显诱导其凋亡.大剂量EGF对LFP培养的HepG2细胞增殖虽有一过性刺激作用,但并不能逆转其抑制作用.LFP抑制HepG2细胞生长和增殖的机制可能与其抑制细胞内Cyclin D1、CDK4的表达有关.

Plasma from patients with liver failure inhibits the proliferation of HepG2 cells

AIM:To explore the mechanisms by which plasma from patients with liver failure inhibits the proliferation of HepG2 cells and to evaluate whether epidermal growth factor (EGF) can reverse this inhibitory effect.METHODS:Plasma samples were collected from three patients with acute-on-chronic liver failure during plasma exchange therapy and treated with heparin.After HepG2 cells were cultured in medium containing 50% plasma from patients with liver failure with or without EGF stimulation,cell proliferation and apoptosis were detected by methyl thiazolyl tetrazolium (MTT) assay and Hoechst staining,respectively.The expression of intracellular cyclin D1 and cyclindependent kinase 4 (CDK4) in HepG2 cells was examined by Western blotting.RESULTS:Treatment with 50% plasma from patients with liver failure for 12 to 72 hours significantly inhibited the proliferation of HepG2 cells in a time-dependent manner when compared to cells cultured with 50% normal control plasma (NCP).EGF at a concentration of 5,10 or 20 g/L significantly induced the proliferation of HepG2 cells cultured with NCP,while only high-dose EGF (20 g/L) showed a transient promotion to the proliferation of HepG2 cells cultured with plasma from patients with liver failure.After stimulation with 20 g/L EGF,the proliferation was still significantly inhibited in cells cultured with patient plasma compared to those cultured with NCP.The presence of 50% patient plasma did not significantly alter apoptosis index of HepG2 cells (P > 0.05).The expression of intracellular cyclin D1 and CDK4 in HepG2 cells was obviously inhibited after treatment with patient plasma for 12 to 72 hours.CONCLUSION:Plasma from patients with liver failure inhibits the proliferation of HepG2 cells possibly by down-regulating the expression of intracellular cyclin D1 and CDK4.EGF can not reverse this inhibitory effect.