CAJ | 학술논문

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。
위료사선초어간세포지방변성적최가유도제급농도,병초보분석지방유제(lipid emulsions, LE)인기초어간세포지방변성적작용궤리,이초어(Ctenopharyngodon idellus)정상간세포위연구대상,건립초어지방변성간세포모형,이함10%태우혈청적기출배양액위대조조,처리조위함20%지방유제0.5~2 mL/L화함20%、50%태우혈청적유도배양액,부육초어간세포48 h 후,정량분석간세포내적감유삼지(TG)함량,관찰지적적취정황급간세포초미결구적변화,검측세포배양상청중곡병전안매(alanine transaminase, ALT)、곡초전안매(aspartate transaminase, AST)적활성, qRT-PCR기술검측지대사관건기인(PPARa、PPARg、SREBP-1c、LPL、Lep화UCP2)적전록수평변화,단백질인적기술검측PPARg、SREBP-1c적단백수평변화。결과발현,함1~2 mL/L LE적유도액조화함20%、50%FBS적유도액조여대조조상비TG함량균현저상승(P<0.05),차20%FBS화각농도LE유도조적전안매활성여대조조상비차이불현저(P>0.05),표명함1~2 mL/L LE적유도액화함20% FBS적유도액균가건립초어영양성지방간세포모형。재간세포지변모형중, PPARγ화 LPL 등지대사기인적표체량현저승고(P<0.05),이 Lep 기인표체량현저강저(P<0.05), PPARγ화SREBP-1c적단백수평승고。결론인위：채용1~2 mL/L LE화20%적FBS균가이재단시간내건립초어간세포지방변성모형,함1 mL/L LE적유도액유도효과최가；간세포내지질적축적가능여지방대사관건기인PPARγ、SREBP-1c、LPL급Lep등밀절상관。
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.