草鱼肝细胞脂变模型的建立及脂代谢基因表达分析

Establishment of a model of grass carp hepatocyte steatosis and analysis of lipid metabolism gene expression

为了筛选草鱼肝细胞脂肪变性的最佳诱导剂及浓度,并初步分析脂肪乳剂（lipid emulsions, LE）引起草鱼肝细胞脂肪变性的作用机理,以草鱼（Ctenopharyngodon idellus）正常肝细胞为研究对象,建立草鱼脂肪变性肝细胞模型,以含10%胎牛血清的基础培养液为对照组,处理组为含20%脂肪乳剂0.5~2 mL/L和含20%、50%胎牛血清的诱导培养液,孵育草鱼肝细胞48 h 后,定量分析肝细胞内的甘油三酯（TG）含量,观察脂滴积聚情况及肝细胞超微结构的变化,检测细胞培养上清中谷丙转氨酶（alanine transaminase, ALT）、谷草转氨酶（aspartate transaminase, AST）的活性, qRT-PCR技术检测脂代谢关键基因（PPARa、PPARg、SREBP-1c、LPL、Lep和UCP2）的转录水平变化,蛋白质印迹技术检测PPARg、SREBP-1c的蛋白水平变化。结果发现,含1~2 mL/L LE的诱导液组和含20%、50%FBS的诱导液组与对照组相比TG含量均显著上升（P〈0.05）,且20%FBS和各浓度LE诱导组的转氨酶活性与对照组相比差异不显著（P〉0.05）,表明含1~2 mL/L LE的诱导液和含20% FBS的诱导液均可建立草鱼营养性脂肪肝细胞模型。在肝细胞脂变模型中, PPARγ和 LPL 等脂代谢基因的表达量显著升高（P〈0.05）,而 Lep 基因表达量显著降低（P〈0.05）, PPARγ和SREBP-1c的蛋白水平升高。结论认为：采用1~2 mL/L LE和20%的FBS均可以在短时间内建立草鱼肝细胞脂肪变性模型,含1 mL/L LE的诱导液诱导效果最佳；肝细胞内脂质的蓄积可能与脂肪代谢关键基因PPARγ、SREBP-1c、LPL及Lep等密切相关。

To establish a model of grass carp （Ctenopharyngodon idellus） hepatocyte steatosis and investigate a possible mechanism for lipid emulsion （LE）-induced steatosis in grass carp hepatocytes, normal grass carp hepa-tocytes were cultured in different inducing media supplemented with different concentrations of LE or fetal bovine serum （FBS）. LE and FBS were tested to identify the best medium and optimal concentration. Grass carp hepato-cytes were cultured in inducing medium containing 20%or 50%FBS, or different concentrations （0.5–2 mL/L） of clinical vein nutrition drug （20% LE） for 48 h. Lipid accumulation was observed by phase-contrast microscopy, the ultrastructure of fatty degeneration of hepatocytes was observed by transmission electron microscope, the triglyceride （TG） content was determined by Oil red O extraction, the activity of ALT and ASL enzyme were ex-amined biochemically, and the expression of the lipid metabolism genes PPARa, PPARg, SREBP-1c, LPL, Lep and UCP2 was determined by real-time PCR. The results revealed that a model of grass carp hepatocyte steatosis can be established using induction medium containing 1–2 mL/L LE, or 20%FBS. The TG level was greatly increased in cells treated with 1–2 mL/L LE, and 20%or 50%FBS compared with that of the control cells （P〈0.05）. How-ever, there was no significant difference in the aminotransferase activity between cells treated with 20%FBS and cells treated with various concentrations of LE（P〉0.05）. In the model of grass carp hepatocyte steatosis, expres-sion of PPARgand LPL genes significantly increased（P〈0.05）, while Lep gene expression decreased sharply （P〈0.05）. In conclusion, our grass carp hepatocyte steatosis model was established over a short time using 1–2 mL/L LE or 20% FBS. Lipid accumulation in hepatocytes was closely related to the expression of lipid metabolism genes （PPARs, SREBP-1c, LPL and Lep）. This study provides the foundations for developing an animal model to explore nutrient metabolism in fish liver, and also provides an alternative way to uncover the mechanism（s） un-derlying metabolic diseases involving lipids.