过表达apoA-Ⅰ对BEL-7402细胞内脂质堆积的影响

Effects of overexpression of apolipoprotein A-Ⅰ on lipid accmulation in BEL-7402 cells

目的通过建立过表达载脂蛋白A-Ⅰ(apolipoprotein A-Ⅰ,apoA-Ⅰ)的细胞模型,用不同浓度及不同类型的脂肪酸作用于细胞,检测过表达apoA-Ⅰ与正常细胞内脂质水平,来研究利用过表达apoA-Ⅰ促进ATP结合盒转运子A1(ATP-bindingcassette transporter A1,ABCA1)介导的脂质转运途径,降低脂质合成,进而影响非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)细胞的脂质堆积的分子机制,探索一种新方法,通过apoA-Ⅰ/ABCA1这一途径改变细胞内脂质代谢来预防NASH。方法①通过不同浓度和类型的脂肪酸处理人肝癌细胞系BEL-7402细胞,作用6、12、24 h后,经油红O染色,检测不同时间脂质在细胞内的定位,从而确定出适合的脂肪酸浓度及作用时间;②通过MTT法检测不同浓度及类型的脂肪酸作用细胞6、12、24 h后,细胞活性的变化情况;③分别转染空载体质粒pcDNA 3.0与质粒pcDNA3.0/apoA-Ⅰ,通过Western blotting的实验方法,检测细胞内、外apoA-Ⅰ的表达情况;④对正常或过表达apoA-Ⅰ的BEL-7402细胞模型,以最适的实验条件作用于正常(对照组)和过表达apoA-Ⅰ(实验组)的BEL-7402细胞,检测细胞内胆固醇、游离脂肪酸以及三酰甘油的含量。结果①200μmol/L或400μmol/L浓度脂肪酸作用6、12及24 h均可使脂质在BEL-7402细胞胞质中堆积,脂质的产生对脂肪酸呈剂量和时间依赖性;②饱和脂肪酸棕榈酸使细胞活性降低,且对细胞生长的抑制作用随棕榈酸的浓度及作用时间的增加而加强;③apoA-Ⅰ过表达于BEL-7402细胞内,且apoA-Ⅰ可以释放到细胞外,协助ABCA1转运胆固醇;④与正常的BEL-7402细胞相比,过表达apoA-Ⅰ组细胞内的三酰甘油、游离脂肪酸以及胆固醇均有显著减少。结论与正常组相比,过表达apoA-Ⅰ可以减少胆固醇、游离脂肪酸及三酰甘油在人肝细胞内的聚积,从而对于NASH的防治具有治疗作用。

Objective To explore a new therapy to change the lipid metabolism and prevent non-alcoholic steatohepatitis（NASH）,to establish a cell model that overexpresses apolipoprotein A-Ⅰ（apoA-Ⅰ）,which promotes cellular cholesterol efflux by interacting with ATP-binding cassette transporter A1（ABCA1）.The effect and its molecular mechanism of apoA-Ⅰ overexpression on cellular lipid levels were evaluated in lipid-loaded cells to investigate the therapeutic effect of apoA-Ⅰ on NASH.Methods ① Human liver cancer cell line BEL-7402 were treated with different concentrations and types of fatty acids for 6,12 and 24 h,cellular lipid accumulation was determined by Oil Red O staining,and optimal concentration and time of fatty acid treatment were then established;② BEL-7402 cells were treated with different concentrations and types of fatty acids for 6,12 and 24 h,and cell activity was determined by MTT;③ Control plasmid and pcDNA3.0/apoA-I plasmid were transfected into BEL-7402 hepatocytes,and intracellular and extracellular apoA-Ⅰ protein levels were determined by Western blotting;④ Cellular cholesterol,free fatty acid and triglyceride content of control cells and apoA-Ⅰ-overexpressive cells were determined respectively.Results ① The lipid accumulation in BEL-7402 cells could be achieved by treatment with 200 μmol/L or 400 μmol/L fatty acids for 6,12 and 24 h,and was both dose-and time-dependent.② Saturated fatty acid palmitic acid decreased the cell activity,and the inhibitory effect of cell growth is coming with increasing time and concentration of palmitic acid.③ ApoA-Ⅰ can be overexpressed intracellularly and excreted extracellularly to facilitate ABCA1 to mediate the transport of cellular cholesterol and phospholipids.④ The cholesterol,free-fatty acid and triglyceride content in apoA-Ⅰ-overexpressive cells were less than that in control BEL-7402 cells.Conclusion Compared with the control cells,the lipid accumulation including cholesterol,free-fatty acid and triglyceride in human hepatocytes was markedly down-regulated by apoA-Ⅰ overexpression.These findings suggest that apoA-Ⅰ overexpression may increase ABCA1-mediated lipid transport,and may have a therapeutic effect on NASH.