| IntroductionHepatic fibrosis is the results of an excessive accumulation of extracellular matrix (ECM) components, due to increased synthesis and decreased degradation. Hepatic stellate cells are generally believed to be a major sourse of collagen and other ECM. In the course of ECM degradation, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) and plasminogen activator inhibitor - 1 ( PAI-1) are important enzymes. Transforming growth factorpi (TGF1) is intense fibrogenic factor and platelet - derived growth factor (PDGF) is the most potent mitogen for HSCs. At present, it is generally thought that HSCs play an important role in alcoholic liver disease ( ALD). Compond prescription of traditional medicine with comprehensive pharmacological effects has shown its special superiority during treatment of hepatic fibrosis. In this study, we observed the effects of KangXian FuFang I ( KXI) on the cell proliferation, collagen sythesis, the expression of MMPs, TIMPs and PAI -ImRNA and secretion of TGFpj and PDGF of rat HSCs pretreated by acetalde-hyde in order to study mechanisms of ALD.Materials and MethodsMaterials :1. Animal, Wistar male rats; collagenase type IV, pronase, Nycodenz were purchased from Sigma, MTT from Fluka; 40% acetaldehyde from Shanghai chemicals company; Anti - Desmin antibody and SP immunohistochemical kitcame from MaiXin in Fujian, radioimmunoassay kit and ELISA kit from the Institute of Shanghai Navy Army and Senxiong company in Shanghai. RT-PCR kit was obtained from Takara Company. PCR primers were from Augct Company in Beijing.2. Preparation of the drug serum: The rats were intragastric infusion with the concentrated oral reagent of KXI and normal saline. The drug serum and control serum were collected in the sterile condition.3. E - Liza Mat -300enzyme - link device, PTC-100 PCR amplifer and 1D Kodak image system: American. JC - 1200 radioimmunoassay device: China Technique University.Methods:1. Isolation, culture and identification of rat HSC:HSCs were obtained from the normal rat liver though perfusion and digestion in situ with pronase and collagenase type IV. Then they were isolated by ul-tracentifugation by gradient media, 18% Nycodenz. HSCs were plated onto un-coated plastic dishes at about density of 1 106 cells/ml and cultured in DMEM. The experiments described in this paper were performed between passage 2 and 4. Immunocytochemical staining for identification of HSC was carried by SP method using anti - Desmin antibody.2. Detection of cell proliferation:Apply the MTT chromometry method. HSCs were suspended in the 20% bovine serum DMEM at a density of 1 105 cells/ml and added into 96 wells plate. (0. 1ml/well). After 72h, three groups were classified: control group, acetaldehyde group and drug serum added into acetaldehyde group. The concentrations of acetaldehyde were 100M, 200M, 300M, respectively. After 24h, add MTT 201 under 570nm wavelength read the light absorb value.3. Measurement of PCIII, LN and HA in conditioned mediaHSCs were plated on the flask (25cm2) at 1+106/flask. After confluence, HSCs were classified into three groups ( equal to method 2) and stimulated by 100 100M acetaldehyde and 10% drug serum. After 24h, the conditioned media were collected. The experiments were performed using radioimmunoassayaccording to instructions.4. Detection of TGF1 and PDGF contents in conditioned mediaHSCs were treated and conditioned media were collected according to method 3. Apply ELISA method and performed according to the protocol.5. Detection of al (I) , 1 (IV) collagen, MMP -1,2,9, TIMP -1,2 and PAI - ImRNA expression by RT - PCRHSCs were treated according to method 3. The total RNA was extracted at 6h and 24h and reversely transcribed into cDNA. PCR was carried on PCR amplifier, b-actin was acted as control gene. PCR reaction conditions and products were different. PCR products were put on 2% agrose gel and electrophore-sis.6. StatisticsData were expresse...
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