乙醇对小鼠原代肝细胞脂肪酸氧化速度的影响.doc

- HE20mmol/L3HC16:0 95%48h20mmol/L40% 3H The effect of ethanol on fatty acid oxidation of mice primary hepatocytes Abstract Objective To isolate the mice primary hepatocytesand the cells were cultured in vitro.The influence of ethanol on fatty acid -oxidation velocity was measured.Methods The mouse was anesthetized with pentobarbitaland the liver was degested with collagenase.The primary hepatocytes were inoculated in collagen-coated dishes.The morphology of cultured primary hepatocytes was observed by HE staining.The fatty acid -oxidation was measured using 910-3H palmitic acid (C16:0) as a tracer.Results The primary hepatocytes were isolated and cultured successfullyand the viability was more than 95%.20mmol/L ethanol could reduce the speed of fatty acid -oxidation about 40%and lead to the increased lipid level in hepatocytes.Conclusion We isolated and cultured the mouse primary hepatocytes in vitroand the influence of ethanol on fatty acid -oxidation was determined.This study is helpful for the research on the lipid metabolism in liver. Key words ethanol;hepatocyte;fatty acid 1 1.1 C57BL/6Sigma910-3HPalmitic acidAmersham BioscienceHisafe 3PE!-empirenews.page- 1.2 C57BL/6100mg/kg3211mM EGTA0.1%Kreb-Ringer20cm4ml/min20ml0.5mg/ml5mM CaCl210minKreb-Ringer10mM70m10%FBSDMEM250g 1.3 4%1210%100u/mlDMEM5%CO237C 1.4 14%OOil Red O2-HE- 1.5 1122 105/12hPBS2500l0.5mg/ml BSA22M 910(n)-3H (1Ci/)Hanks20mM337C2h0.5ml Hanks2Folch8ml /(2:1)0.5ml 2 M KCl/2 M HCl30min/10ml3H2OBrad-ford 2 2.1 95% 2.2 12h2448h1A>41B1C1 Fig 1 the morphology of cultured primary hepatocytesA: Cultured 24 hr; B: Cultured 96 hr; C: the division the hepatocytes 2.3 3.12nmol/mg protein/h20mmol/L1.76pmol/mg protein/h40%2!-empirenews.page-2 Fig 2 The effect of ethanol to the rate of fatty acid oxidation of cultured hepatocytes 2.4 O20mmol/L6h33 20mmol/L6hO40Fig 3 The Oil Red O staining of hepatocytes treated 6h with 20mmol/L ethanol (40)A: The hepatocytes without treating with ethanol; B: The hepatocytes treated with 20mmol/L ethanol. 3 alcoholic liver diseaseALDINADINADHNAD/NADH(reactive oxygen speciesROS) oxidative stressOS2412 57reduced glutathioneGSHHydroxyl Radical1-1-H