| Background and purpose:Recently, along with the increasing alcohol wastage,the incidence of alcoholic liver disease is increasing too. Long-term consumption of alcohol result in spectrum of liver abnormalities, rangiing from simple fatty liver(steatosis) to steatohepatitis, cirrhosis, and hepatocellular carcinoma. In the United States, approximately 10 to 35 percent of heavy drinkers develop alcoholic hepatitis, and 10 to 20 percent developp cirrhosis. Approximately 10,000 to 24,000 deaths from cirrhosis may to be attributable to alcohol consumption each year.Liver fibrosis is a dynamic state between matrix production and degradation, and result in excessive deposition of newly-formed ECMcomponents. Interstitial collagenase(MMP-l,MMP-8,MMP-13) has been considered an essential enzyme for collagenolysis in liver fibrosis, because type I and HI collagen, which increases predominantly in liver fibrosis, cannot be degraded by other MMPs such as MMP-9 or MMP-3. Hepatic fibrogenesis is not caused solely by an increase in connective tissue synthesis, but is basically the result of an imbalance between enhanced matrix synthesis and diminished or alterde matrix breakdown. Recent studies have indicated that a remarkable increase of MMP-13 mRNA in the early stage of rat liver fibrosis induced by chronic CC14 intoxication, but once the fibrosis became prominent, the faint band for MMP-13 mRNA was detected only by RT-PCR, and only few studies about MMP-13 in alcoholic liver disease has been reported. Vascular endothelial growth factor(VEGF) is an important endothelial cell mitogen that induced endothelial proliferation, angiogenesis, and capillary hyperpermeability. Identification of VEGF both in its protein level and mRNA level and its significance have been extensively studies in various hepatic disease. It has been testified that VEGF plays an important role in the growth of sinusoidal endothelial cells and hepatocytes, or termed liver regeneration, and in the development of primary and secondary liver carcinoma and benign liver pathological changes such as hemangioma, focal hyperplasis, and hepatitis. However the role of VEGF in ALD has not been elucidated.The present study aimed to demonstrate the gene expression of MMP-13 and VEGF by reverse transcription-polymerase chainreaction(RT-PCR) in the progressive phases of ethanol induced experimental liver fibrosis in rats, and revealed that MMP-13 and VEGF participate in the mechanism of alcoholic liver disease.Material and Methods:Thirty-four healthy weighing 120-210g S-D rats were used. Among them, twenty-four rats were given alcohol (56%, 8g/kg) every day. Those rats were named alcohol group. Other rats were given equality normal saline as control group. We took out some rats from alcohol group at random at 4, 12, 24 weeks, collected the liver of each rats to study. The part of the resected liver samples were immediately fixed in 10% formaldehyde solution, the other we're frozen-80癈.Histological examination was performed using Masson staining to observe hepatic fibrosis degree. And SSS semi-quantitative scoring method was used to analyze those histology change. Total RNA was isolated from control and ethanol-treated rat liver using Trizol reagent. And total RNA was reverse transcribed with 5 units of Avian myeloblastosis virus reverse transctiptase(GIBCOBRL). Following complementary DNA (cDNA) synthesis, gene expression of MMP-13 and VEGF were determined by amplification of cDNA. MMP-13 primer(355bp): sense sequence 5' -TGA CTA TGC GTG GCT GGA A-3 ', and the antisense sequence 5' -AAG CTG AAA TCT TGC CTT GGA-3 '. VEGF primer(568bp): sense sequence 5' -TGC ACC CAC GAC AGA AGG GGA-3 ', and the antisense sequence 5' -TCA CCGCCT TGG CTT GTC ACA T-3 '. As a positive control for each RNA prepartion, the glyceraldehyde 3-phosphate dehydrogenase(GAPDH) sequence(349bp) was also amplified in separate tubes using sense 5' -CAT GOT CTA CAT GTT CCA GT-3 ' and antisense 5' -GGC TAA GCA GTT GGT GOT GC-3 'primers...
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