非酒精性脂肪性肝病风险基因SIRT3的关联验证和体内过表达效应研究

目的 研究肝脏脂肪酸氧化关键分子SIRT3编码基因变异与非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)发生的关系,并在小鼠体内研究人源SIRT3保护性等位型基因过表达对脂肪肝相关表型的影响.方法 建立非酒精性脂肪肝的人群研究队列,提取基因组DNA对编码区基因变异rs11246020进行基因分型,并与疾病发病与否做关联分析;建立小鼠NAFLD模型,在小鼠肝脏过表达人源SIRT3基因,8周后检测小鼠肝脏相关生化指标及肝脏HE染色、油红O染色情况,并提取RNA和蛋白采用Real-time PCR和Western blot检测Pparα、Acox1、cpt1a、cpt1b和SIRT3的转录或表达水平.结果 与对照组相比,SIRT3基因编码区错意突变rs11246020的GA+AA基因型在NAFLD组分布较低,而GG基因型分布较高,差异具有统计学意义(P＜0.05);在小鼠肝脏过表达人源SIRT3的A等位型基因后,肝脏脂肪蓄积降低,肝细胞空泡化程度降低,肝脏和血清游离脂肪酸以及肝脏甘油三酯、总胆固醇水平显著降低(P＜0.05),肝脏脂肪酸代谢相关基因Pparα和Acox1基因转录水平降低(P＜0.05).同时,小鼠体质量上升被有效控制(P＜0.05).结论 SIRT3基因变异rs11246020与NAFLD风险相关,小鼠体内过表达SIRT3可显著改善脂肪酸代谢和肝脏脂肪蓄积.

Association of SIRT3 gene variation with risk of non-alcoholic fatty liver disease and functional study of the gene in a mouse model

Objective To investigate the association between silent mating-type information regulation 2 homolog 3 (SIRT3) gene variation and the risk of non-alcoholic fatty liver disease (NAFLD) and explore the gain-of-function effect of SIRT3 in a mouse model of NAFLD.Methods Blood samples were collected from 374 patients with NAFLD and 372 health control subjects at the Health Examination Center of Southwest Hospital from March 2014 to March 2015.Genome DNA was extracted from the samples for rs11246020 genotyping using Mass-Array system,and the genotype distribution of rs11246020 was analyzed.A plasmid carrying human SIRT3 gene was transfected in a mouse model of NAFLD,and in 8 weeks later,the biochemical indexes and levels of inflammatory cytokines in the serum and liver of the mice were evaluated;the pathology of the liver tissues was observed with HE and oil Red O staining,and the expression levels of Pparα,Acox1,cpt1a,and cpt1b mRNAs and SIRT3 protein in the liver were detected using real-time PCR and Western blotting.Results Compared with the control group,the patients with NAFLD showed a significantly decreased frequency of GA + AA genotype and an increased frequency of GG genotype of rs11246020 in SIRT3 gene (P ＜ 0.05).In the mouse model of NAFLD,over-expression of human SIRT3 in the liver significantly reduced the levels of total cholesterol,triglyceride,and free fatty acids in both the serum and the liver (P ＜ 0.05) and obviously lowered the expression levels of Pparα and Acox1 mRNAs in the liver (P ＜ 0.05).Pathological examination of the liver tissue from the mice with SIRT3 over-expression revealed significantly reduced accumulation of fat vacuoles and lipid droplets.SIRT3 over-expression also effectively controlled the weight gain in the mice (P ＜ 0.05).Conclusion SIRT3 gene variation is associated with the risk of NAFLD,and SIRT3 over-expression improves NALFD in mice possibly by modulating fatty acid metabolism.