Stargardt病Ⅲ型致病基因ELOVL4在脂肪酸代谢中的功能研究

Role of Stargardt type m macular dystrophy gene ELOVL4 in fatty acids metabolism

背景研究证实，超长链脂肪酸延伸酶4（ELOVIA）基因是Stargardt病的致病基因，且ELOVIA可能是c≥28的超长链脂肪酸的延伸酶，而常染色体显性遗传Stargardt病Ⅲ型（STGD3）的发病可能与视网膜中c≥28的超长链脂肪酸的代谢有关。探讨ELOVL4在STGD3发病中的作用及其与超长链脂肪酸代谢的关系有望对STGD3的治疗提供新的思路。目的探讨ELOVIA基因在STGD3发病和进展中的作用及机制。方法将腺病毒（Ad）转染重组病毒质粒36h的人胚肾293细胞（HEK293）以纯化病毒，构建携带ELOVIA基因和绿色荧光蛋白（GFP）的重组腺病毒载体并转人培养的大鼠肾上腺嗜铬细胞瘤细胞株（PCI2），将培养的PCI2细胞分成PCI2组、PCI2＋Ad5一GFP组和PCI2＋Ad5一ELOVL4组，根据分组情况分别将Ad-GFP和Ad-ELOVL4病毒质粒按1×10～2×10。噬斑形成单位（pfu／m1）的滴度加入含质量分数2％胎牛血清（FBS）的细胞培养液转导24h，采用实时定量PCR法（qRT—PCR）定量分析各组细胞中ELOVIAmRNA的相对表达量；采用Westernblot法检测细胞中ELOVL4蛋白的表达。分别在各组培养基中加入花生四烯酸（AA或20：4n6）、二十碳五烯酸（EPA或20：5n3）和二十二碳六烯酸（DHA或22：6n3），孵育48h后进行脂肪酸提取，通过气相色谱一质谱联用仪（GC-MS）分析各组细胞中超长链脂肪酸产物。结果PCI2＋Ad-ELOVL4组、PCI2＋Ad-GFP组和PCI2组间细胞中ELOVL4mRNA表达水平（ELOVIAmRNA／RPLl9mRNA）分别为0．833±0．138、0．027±0．002、0．024±O．002，3个组的总体差异有统计学意义（F=102．700，P=0．000），其中PCI2＋Ad5一ELOVL4组PCI2细胞中ELOVIAmRNA表达水平是PCI2组和PCI2＋Ad5-GFP组的30～40倍；Westernblot法检测结果证实，PCI2＋Ad5-ELOVIA-组PCI2细胞中ELOVL4蛋白呈阳性表达。GC-MS法检测发现PCI2＋Ad5-ELOVL4组PCI2细胞中有C28-C38多不饱和脂肪酸的合成，其中均以C34和C36多不饱和脂肪酸合成量最多。结论ELOVL4蛋白酶可催化C28-C38多不饱和脂肪酸的合成，这个结论为STGD3患者的治疗提供了新的思路。

Background Researches determined that ELOVIA gene is a disease-causing gene of Stargard tmacular dystrophy and is a elongation enzyme of very long chain fatty acids. Stargardt type Ⅲ （ STGD3 ） may be associated with the metabolism of extensive enzyme of very long chain fatty acids. To explore the effect of ELOVL4 in STGD3 and its relationship with the metabolism of extensive enzyme of very long chain fatty acids is of important clinical significance. Objective This study was to determine the role of ELOVL4 gene for the pathogenesis of STGD3. Methods Recombinant adenovirus type 5 carrying mouse ELOVL4 gene and green fluorescent protein （GFP） was transfected into pheoehromocytoma cells （PC12 cells） ,and then the cells were divided into PCI2 group, PC12＋Ad5-GFP group and PC12＋Ad5-ELOVL4 group. Ad-GFP or Ad-ELOVL4 was added into the culture medium for 24 hours with the virus plasmid 1 x 104-2x 104 pfu/ml. Expression of ELOVL4 mRNA in the PC12 was quantified by quantitative real time PCR（qRT-PCR） and was described as relative value to the expression of RPL19. ELOVIA protein was assayed by Western blot. The transfected ceils were treated with arachidonic acid （ AA, 20：4n6 ） , eicosapentaenoie acid （ EPA, 20 ： 5n3 ） and docosahexaenoie acid （ DHA, 22 ： 6n3 ） individually for 48 hours. Thecells were collected,and total lipids were extracted, and fatty acid methyl esters were prepared and analyzed by gas chromatography-mass spectrometry （ GC-MS）. Results The relative expression levels of ELOVIA mRNA in PC12 ceils in the PC12＋Ad5-ELOVIA group,PC12＋Ad5-GFP group and PC12 group were 0. 833＋ 0. 138,0. 027＋0. 002 and 0. 024±0. 002, with a significant difference among the 3 groups （F = 102. 700, P = 0. 000） , and relative expression levels of PC12＋Ad-ELOVIA were 30-40 folds more than those in the PC12 group and the PC12＋Ad-GFP group. Western blot assay showed a stronger response band for ELOVIA protein in the PC12＋Ad-ELOVIA group. GC-MS found that abundant polyunsaturated fatty acids （C28-C38） were synthesized by PC12 cells in the PCI2＋Ad-ELOVIA group,with the more levels in C34 and C36. Conclusions ELOVL4 can promote the synthesis of C28-C38 polyunsatured fatty acid in PC12 cells,which offers a novel clue for the treatment of STGD3.