Identification of HGF indncing factor in serum from patients with Liver diseases

将MRC-5细胞接种于24孔塑料培养皿中，加入0.5 ml添加10%FCS和抗生素的DMEM，在37 ° C、5%CO_2、有或无患者血清的潮湿环境中培养。血清标本取自正常人、暴发性肝衰竭（FH）、急性肝炎（AH）、慢性肝炎（CH）、肝硬化（LC）和肝细胞癌（HCC）患者。将血清样品以5%的终浓度加入培养基中，并在加入样品后培养细胞24小时。用酶联免疫吸附试验（ELISA）测定培养上清中肝细胞生长因子（HGF）的含量，并以单位细胞蛋白的HGF含量（ng/mg蛋白）表示。AH和CH患者血清中HGF含量与血清中HGF含量呈正相关。北方印迹分析显示，与AH患者血清一起培养的MRC-5细胞的HGF mRNA水平增加。热处理（56 ℃，30分钟）和胰蛋白酶或糜蛋白酶处理可使患者血清中的HGF诱导活性消失。我们还检测了白细胞介素-1（IL-1）、白细胞介素-2（IL-2）、肿瘤坏死因子-α（TNF-α）、佛波醇酯（PMA）、地塞米松和肝素对用具有高HGF诱导活性的血清培养的MRC-5细胞中HGF产生的影响。IL-1、TNF-α和PMA对患者血清刺激MRC-5细胞产生HGF有相加作用，而地塞米松则抑制HGF的产生。

MRC-5 cells were plated on 24-well plastic dish in 0.5 ml of DMEM supplemented with 10% FCS and antibiotics, and cultured at 37 ﾟC in a humidified atmosphere of 5% CO_2 in the presence or absence of patient's serum. Serum samples were obtained from normal subjects, patients with fulminant hepatic failure (FH), acute hepatitis (AH), chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Serum samples were added to the culture medium at a final concentration of 5% and the cells were cultured for 24 hours after the addition of samples. Hepatocyte growth factor (HGF) in the culture supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) and HGF production was expressed as HGF amount per cellular protein (ng/mg protein).Sera from patients with AH and CH increased HGF production in MRC-5 cells with dose- and time-dependent manner. The relationship between HGF production by patients' serum and HGF levels in serum of AH and CH patients was positively significant. Northern blot analysis revealed an increase in HGF mRNA levels of MRC-5 cells cultured with serum from AH patients. HGF-inducing activity in the patients' serum was disappeared by heat treatment (56ﾟC,30min) and treatment with trypsin or chymotrypsin. We also examined the effects of interleukin-1 (IL-1), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), phorbol ester (PMA), dexamethasone and heparin on HGF production in MRC-5 cells cultured with serum having a high HGF- inducing activity. IL-1, TNF-alpha and PMA had an additive effect on HGF production stimulated by patient's serum in MRC-5 cells, however, dexamethasone suppressed the HGF production.In summarize, HGF-inducing activity was present in serum of patients with various liver diseases and this activity may be a physical HGF inducer in humans.