脂肪酸影响草鱼肝细胞脂质蓄积状态及诱导其凋亡的离体研究

INFLUENCE OF FATTY ACIDS ON LIPID ACCUMULATION AND APOPTOSIS STATUS OF GRASS CARP CTENOPHARYNGODON IDELLUS HEPATOCYTE IN VITRO

为探究外源脂肪酸对草鱼肝细胞脂质代谢及健康状况的影响及其机理,体外培养草鱼肝细胞,并采用不同浓度(0—1 mmol/L)油酸(Oleic acid)进行细胞孵育,噻唑兰比色法(Methyl thiazolte trazoliu,MTT)和油红O染色提取法检测肝细胞活力及脂质蓄积状况,BODIBY和DAPI染色法观察肝细胞脂滴及细胞核情况,流式细胞术检测肝细胞凋亡率变化,Real-time q PCR检测脂质合成标志基因过氧化物酶体增殖物激活受体γ(Peroxidase proliferation activated receptor,PPARγ)和CCAAT/增强子结合蛋白α(CCAAT/enhancer binding protein alpha,C/EBPα)、凋亡相关基因Caspase家族等的表达情况。结果显示,随着油酸处理浓度的增加,肝细胞活力和细胞内脂质积累呈现先上升后下降的趋势,分别在0.4和0.6 mmol/L时达到最大值(P<0.05);肝细胞凋亡率则先下降后上升,在0.4 mmol/L油酸处理时最低,1 mmol/L油酸处理时最高(P<0.05);此外,0.4 mmol/L油酸处理抑制了肝细胞Caspase-3b和Caspase-9基因的表达,上调Bcl-2/Bax m RNA比值(P<0.05),而0.8 mmol/L油酸处理显著促进Caspase-3b、Caspase-8、Caspase-9及凋亡诱导因子(Apoptosis inducing factor,AIF)基因的表达,下调Bcl-2/Bax的m RNA比值(P<0.05)。研究表明,一定浓度的脂肪酸可增强草鱼肝细胞活力,促进胞内脂质积累,抑制细胞凋亡,而脂肪酸浓度过高则抑制肝细胞活力并诱导肝细胞凋亡,其作用与脂肪酸影响脂质代谢及凋亡基因的表达有关。

To investigate the effects of fatty acids on hepatocyte lipid metabolism and health status of grass carp （Cte-nopharyngodon idellus）, the normal grass carp hepatocytes were cultured with different concentrations of oleic acid （0—1 mmol/L） for 24h to measure cell viability and lipids content by methylthiazolyl tetrazolium （MTT） assay and oil red O staining, respectively. The lipid droplets and nuclear of hepatocytes were observed with BODIPY and DAPI staining. Hepatocyte apoptosis was analyzed by flow cytometry. Real-time qPCR was performed to detect the mRNA expression of peroxisome proliferator-activated receptorγ（PPARγ）, CCAAT/enhancer binding protein alpha （C/EBPα） and apoptosis related genes [Caspase3a, Caspase-3b, Caspase-8, Caspase-9, apoptosis inducing factor （AIF）, Bcl-2, Bax]. The results showed that the cell viability and intracellular lipid accumulation of hepatocytes increased signifi-cantly after treatment with 0.6 and 0.4 mmol/L oleic acid （P〈0.05）. The lowest hepatocytes apoptosis rate was in 0.4 mmol/L group and the highest was in 1 mmol/L oleic acid group （P〈0.05）. The mRNA levels of Caspase-3b and Caspase-9 reduced significantly and the mRNA ration of Bcl-2/Bax was elevated by 0.4 mmol/L oleic acid （P〈0.05）. Consistently, 0.4 mmol/L oleic acid significantly reduced caspase-3b, caspase-9 and AIF mRNA levels and increased the mRNA ratio of Bcl-2/Bax （P〈0.05） and these genes showed opposite expression pattern in hepatocytes treated with 0.8 mmol/L oleic acid （P〈0.05）. These results demonstrated dosage-dependent role for fatty acids in grass carp hepato-cytes viability and intracellular lipid accumulation via the regulation of the genes related to lipid metabolism and apop-tosis.