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

摘要:

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(n3 PUFA)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。

Abstract:

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 48h 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. From 20:5n3/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 EPA was 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.