【关键词】 乙醇；肝细胞；脂肪酸
The effect of ethanol on fatty acid oxidation of mice primary hepatocytes
【Abstract】 Objective To isolate the mice primary hepatocytes，and the cells were cultured in vitro.The influence of ethanol on fatty acid β-oxidation velocity was measured.Methods The mouse was anesthetized with pentobarbital，and 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 β-oxidation was measured using ［9，10-3H］ palmitic acid (C16:0) as a tracer.Results The primary hepatocytes were isolated and cultured successfully，and the viability was more than 95%.20mmol/L ethanol could reduce the speed of fatty acid β-oxidation about 40%，and lead to the increased lipid level in hepatocytes.Conclusion We isolated and cultured the mouse primary hepatocytes in vitro，and the influence of ethanol on fatty acid β-oxidation was determined.This study is helpful for the research on the lipid metabolism in liver.
【Key words】 ethanol;hepatocyte;fatty acid
肝脏在脂肪、糖和蛋白质的代谢过程中具有重要的作用。在体内肝细胞的代谢受到各种因素的影响。酒精在人肝细胞中主要由乙醇脱氢酶和过氧化氢酶代谢成具有细胞毒性的乙醛，然后由乙醛脱氢酶代谢为乙酸。在这个过程中，肝脏的糖脂代谢将受到影响。本研究通过体外分离肝细胞和培养，研究乙醇对肝细胞的脂肪酸氧化速度的影响。
1 材料与方法
1.1 材料 C57BL/6小鼠由第四军医大学实验动物中心提供；戊巴比妥钠、胶原酶、胰岛素、异丙肾上腺素，棕榈酸为Sigma公司；［9，10-3H］Palmitic acid为Amersham Bioscience产品；Hisafe 3闪烁计数液为PE公司产品。
1.2 肝细胞的分离 取C57BL/6小鼠，采用戊巴比妥钠（100mg/kg）麻醉；打开小鼠腹腔，向左侧移开小肠，暴露下腔静脉和门静脉，使用3号丝线从肾静脉近端绕过下腔静脉；采用21号套管针从下腔静脉传入，直至接近肝静脉；用丝线固定套管针，采用含有1mM EGTA和0.1%葡萄糖的Kreb-Ringer缓冲液灌注肝脏，同时迅速切断门静脉使灌注液从门静脉流出，灌注压力为20cm水柱，灌注速度为4ml/min；灌注20ml后更换含有0.5mg/ml胶原酶和5mM CaCl2的灌注液继续灌注10min，立刻小心取出肝脏，置于含有Kreb-Ringer缓冲液的10mM培养皿中，用剪刀剪开肝脏包膜，轻轻晃动使肝细胞释放出来；采用70μm的细胞滤器过滤；使用10鸖的DMEM洗涤细胞2次，50×g离心。
1.3 存活性鉴定 分离后的肝细胞立刻用4%台盼蓝染色，在显微镜下观察，核呈蓝色为死细胞，拒染的细胞为活细胞，将细胞接种于胶原包被的12孔培养皿中，采用含有10%胎牛血清，100u/ml青霉素和链霉素的DMEM培养液，在5%CO2，37°C培养。