乙酰辅酶A羧化酶反义寡核苷酸对非酒精性脂肪肝细胞模型的作用

Effect of acyl-CoA carbrolase antisense oligonucleotide on non-alcoholic fatty liver cell model

目的观察乙酰辅酶A羧化酶(ACC)反义寡核苷酸(ASODN)对HL-7702肝细胞脂肪变性的影响。方法用计算机软件设计并合成特异性靶向ACC的硫代修饰ASODN。用静脉脂肪乳剂体外培养正常HL-7702肝细胞48 h,制备体外脂肪肝细胞模型。以脂质体法将ACC-ASODN转染至脂肪肝细胞模型,设置空白组(HL-7702肝细胞组)、模型组(脂肪肝细胞模型组)、低高2个浓度实验组(5,10μmol·L^(-1)ACC-ASODN转染组)。以ACC-ASODN转染入脂肪肝细胞模型24 h后,放射性同位素技术测定ACC酶活性,HPLC法测定丙二酰辅酶A含量,放射性同位素技术测定肉碱棕榈酰转移酶Ⅰ(CPT I)活性,用全自动生化分析仪测定4组肝细胞内三酰甘油(TG)含量。结果 ACC-ASODN转染入脂肪肝细胞模型24 h后,空白组、模型组及低、高2个浓度实验组的肝细胞ACC活性(U)分别是0.74,0.33,0.20,0.18;这4组的细胞内丙二酰辅酶A含量(nmol·L^(-1))分别是14.3,12.4,7.5,6.8;这4组的细胞CPTⅠ活性(U)分别是1.33,1.47,3.85,3.95;这4组的TG含量分别是0.20,0.52,0.37,0.28 mmol·L^(-1)。模型组与空白组的以上几项指标比较,差异均有统计学意义(均P<0.05);实验组与模型组的以上几项指标比较,差异均有统计学意义(均P<0.01)。结论 ACC-ASODN作用于肝细胞,通过抑制肝细胞ACC活性,使TG含量下降,从而减轻肝细胞脂肪变性。

Objective To observe the effect of acyl-CoA carbrolase（ ACC） antisense oligonucleotide（ ASODN） on steatosis of HL-7702 hepatocytes. Methods The specific phosphorothioate modified ACC targeting ASODN were designed and synthesized by a computer software.Normal HL-7702 hepatocytes were cultured in vitro using intravenous lipid emulsions,a large number of triglyceride was induced in hepatocytes within 48 h,and a model of fatty liver cells in vitro was successfully formed. ACC-ASODN was transfected into the hepatocyte model by liposome method. The blank group（ HL-7702 cells）,model group（ fatty liver cells of model） and low and high concentrations of the two experimental groups（ 5,10 μmol·L^（-1） ACC-ASODN） were set up. ACC-ASODN was transfected into fatty liver cell model 24 h later. ACC activity was determined by radioisotope technology. The malonyl coenzyme Acontent was determined by HPLC. The carnitine palmitoyltransferase Ⅰ（ CPT Ⅰ） activity was determined by radioisotope technique. The level of triacylglycerol（ TG） was determined by automatic analyzer. Results The value of the liver cell ACC activity（ U） in blank group,model group and experimental-L,-H groups were respectively 0. 74,0. 33,0. 20,0. 18. The value of the intracellular content（ nmol·L^（-1）） of malonyl CoA in above four groups were respectively 14. 3,12. 4,7. 5,6. 8. The value of CPT Ⅰ（ U） in above four groups were respectively 1. 33,1. 47,3. 85,3. 95; the value of TG conten（ mmol·L^（-1）） in above four groups were respectively 0. 20,0. 52,0. 37,0. 28. Compared with blank group,the factors in model group had significant differences（ all P〈 0. 05）. Compared with model group,the factors in experimental-L,-H groups had significant differences（ all P 〈0. 01）. Conclusion ACC-ASODN acts on hepatocytes,and by inhibiting the activity of ACC,the content of TG is decreased,and the steatosis of hepatocytes is alleviated.