| Promoting hepatocyte growth factor (pHGF) is a kind of active substance extracted from suckling pig liver, which is based on the method of LaBrecque. And its biological characteristics are the same as hepatic stimulator substance (HSS) discovered by LaBrecque from the rats liver after hepatoectomy. For the purpose of further investigation on HSS or pHGF, and on the mechanism of fibrosis, the followings have been done: (1) observing the influence of pHGF on the proliferation of HSF and HSC-T6. (2) studying the effect of pHGF on the extracellular matrix (ECM) secreted by HSF and HSC-T6. (3) investigating the influence of pHGF on the activity of human COL1A1 promoter.Section I The influences of pHGF on the proliferation of HSFandHSC-T6The study of liver fibrosis has long been concentrated on the hepatocytes, hepatic stellate cells (HSC) and Kupffer's cells,etc.It has been reported that pHGF may inhibit the rats liver fibrosis and protect the liver from injury.To observe the functions of pHGF on cells in vitro, we choosed HSF and HSC-T6 as cell models and investigated the influence of pHGF on proliferation of HSF and HSC-T6.HSF were obtained from the skin of a three-year-old baby whoreceived an burn operation, which were cultured on routine strapping method. HSC-T6 were kindly provided by lab of RWTH in Germany.The concentration of pHGF were adjusted to 62.5,125,250,500,1000 u g/ml using culture media. HSF and HSC-T6 in logarithmic growth phase were incubated in culture plates in which the cultured cell density were adjusted to 1 x lOVml.OD values of the cells were tested by the method of MTT after 24hr incubation.The results showed that:(l)pHGF at the group of the concentration of 500 and 1000 u g/ml inhibited the proliferation of HSF significantly vs central group (p<0.001), and pHGF had no significant inhibiting effects at the groups of concentration below 250 u g/ml vs control group (p>0.05). (2) pHGF at all of the groups in different concentrations had the inhibiting effects on the proliferation of HSC-T6 vs central group (p<0.01), and the lowest inhibiting concentration of pHGF was 62.5 H- g/ml. The inhibiting effect of pHGF on HSC-T6 is dose-dependant.Section II The influences of pHGF on the ECM produced byHSF and HSC-T6Extra deposition of extracellular matrix (ECM) in Disse's spaces is the main pathologic basis in liver fibrogenesis. ECM is complicatedly composed of collagen proteins, glycoproteins and proteoglycan etc. The type I and III collagens are the main components in the livers of fibrosis, and account for about 60% and 20% respectively. Hyaluronic (HA) is akind of important glyosaminoglycan (GAG) which can combine to polyproteins with other GAGs. HA may increase highly during liver fibrosis and the ability of clearing HA by endothelial cells decreases at the same time.The sensitivity and specifity of HA in liver fibrosis as an index in blood test have long been accepted. The influences of pHGF on ECM secreted by HSF and HSC-T6 were investigated , and the type I collagen (COL1) and HA were taken as represents of ECM.The culture of HSF and HSC-T6 were performed according to the first section. After 24hr incubation of HSF and HSC-T6 at the density of 1 X 105/ml , the fresh pHGF 2ml were maintained in the culture cells. The concentrations of pHGF were 200 and 400 g/ml. The supernatant fluid of culture cells was collected after 24hr incubation, and then the HA and COL1 in the supernatant fluid were tested by the method of radioimmunoassay. The results showed that: (1) The amounts of FiA secreted by HSF were inhibited markedly by pHGF at the concentrations of 200 and 400 g/ml, and the levels of COL1 secreted by HSF were inhibited by pHGF at the concentration of 400 g/ml vs control group (p<0.05). There was no significantly inhibiting effect on HSF secreting COL1 by pHGF at the group of 200 g/ml comparing with control group.(p>0.05) .(2) pHGF at the group of 200 g/ml had no inhibiting effect on HSC-T6 producing HA vs control group (p>0.05), but had markedly inhibiting effe... |
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