ELOVL4基因过表达对n3和n6超长链多不饱和脂肪酸生物合成效率的影响

Effects of ELOVL4 gene overexpress on the synthesis efficiency of n3 and n6 very long chain polyunsaturated fatty acids

目的:ELOVL4是常染色体显性Stargardt氏黄斑变性的致病基因,ELOVL4蛋白酶参与n3和n6超长链多不饱和脂肪酸(very long chain polyunsaturated fatty acid,VLC-PUFA)的合成,比较两者的生物合成效率,对治疗Stargardt氏黄斑变性有指导意义。方法:构建携带ELOVL4基因和绿色荧光蛋白的重组腺病毒,转入培养的PC12细胞,将细胞分成三组:PC12、PC12+Ad-GFP和PC12+Ad-ELOVL4,前两组为对照组,通过qRT-PCR定量分析ELOVL4基因的表达量,Western Blot检测ELOVL4蛋白的表达;等浓度(1∶1)加入EPA(n3PUFA)和AA(n6 PUFA),孵育48h之后进行脂肪酸提取,通过气相色谱-质谱法(gas chromatography-mass spectrometry,GC-MS)分析超长链脂肪酸的成分。结果:GC-MS检测到分别用EPA和AA处理后的PC12+Ad-ELOVL4的细胞中有n3 VLC-PUFA的表达,34∶5n3和36∶5n3是20∶5n3/EPA的主要产物,分别为0.71%和1.6%;34∶4n6和36∶4n6是20∶4n6/AA的主要产物,分别为0.46%和0.61%;EPA所产生的n3 VLC-PUFAs总和是AA所产生的n6 VLC-PUFAs总和的2倍。结论:在ELOVL4蛋白作用下,EPA合成VLC-PUFAs的效率高于AA,饮食中给予适当比例的n3/n6 PUFAs,可能是治疗STGD3疾病的方式之一。

AIM：To compare the synthesis efficiency of n3 and n6 very long chain polyunsaturated fatty acid（ VLC- PUFA）by overexpressing ELOVL4 protein,providing guidance for treating Stargardt- like macular dystrophy（STGD3）. METHODS： To establish recombinant adenovirus with the ELOVL4 protein and green fluorescent protein,transferred into cultured PC12 cells. The cells were divided into 3 groups： PC12,PC12 ＋ Ad- GFP and PC12 ＋ Ad-ELOVL4,former two groups serve as controls. ELOVL4 gene expression was quantified by qRT- PCRs. ELOVL4 protein was analyzed by Western- Blot（ WB）. The transduced cells were treated with both EPA and AA（1∶ 1）.After 48 h of incubation,cells were collected,total lipids extracted and fatty acid methyl esters prepared and analyzed by gas chromatography- mass spectrometry（GC- MS）.RESULTS： When supplemented together,20∶ 5n3（EPA）and 20∶ 4n6（ AA） were efficiently taken up at almost the same amounts in the PC12 cells regardless of ELOVL4 expression. The ELOVL4- expressing cells elongated both EPA and AA to a series of n3 and n6 VLC- PUFAs. From20∶ 5n3/EPA,34∶ 5n3 and 36∶ 5n3 account for 0. 71% and 1. 6%,respectively. From 20∶ 4n6/DHA,34∶ 4n6 and 36∶4n6 were only 0. 46% and 0. 61%,respectively. The total relative mol% of n3 VLC- PUFAs synthesized from EPAwas almost two times that of n6 VLC- PUFAs synthesized from AA.CONCLUSION： ELOVL4 protein preferentially elongates n3 PUFA to VLC- PUFAs over n6 PUFA. Dietary supplementation of appropriate n3/n6 PUFAs may provide STGD3 patients with some therapeutic benefits.