- 學位論文
探討長鏈脂肪酸去飽和酶及延長酶強迫表現對三種人類細胞株合成二十二碳六烯酸之影響
Effect of long chain fatty acid desaturase and elongase overexpression on docosahexaenoic acid biosynthesis in three human cell lines
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摘要
二十二碳六烯酸 (docosahexaenoic acid, DHA, 22:6n-3) 為n-3多元不飽和脂肪酸，主要富含在腦部神經細胞膜中，對維持神經細胞正常結構及功能十分重要。DHA可以經由前驅物α-次亞麻油酸 （α-linolenic acid, ALA, 18:3n-3），透過去飽和酶 (fatty acid desaturase, FADS)及延長酶 (elongase of very long chain fatty acid, ELOVL)經由一系列去飽和及加長碳鏈後生成。在大腦發育時期，嬰兒有能力自行合成DHA，但成年之後DHA在人體中的合成效率卻很低，因此本論文研究是否因人類細胞中缺乏特定的酵素而導致無法有效合成DHA，又能否透過強迫表現該酵素來克服。 本論文選用了MCF-7、SH-SY5Y及HEK293T三種人類細胞株，加入不同的DHA前驅物ALA、EPA (20:5n-3)、DPA (22:5n-3)、24:5n-3或24:6n-3培養後，以氣相層析儀分析其脂肪酸組成，並對照參與DHA合成相關酵素FADS2、FADS1、ELOVL5及ELOVL2之即時聚合酶鏈鎖反應結果，找出各細胞中限制DHA合成之酵素。 本論文發現MCF-7主要缺乏FADS2；SH-SY5Y主要缺乏ELOVL2；HEK293T則是FADS2及ELOVL2兩種都缺乏，導致無法合成DHA。而當MCF-7強迫表現FADS2、SH-SY5Y強迫表現ELOVL2或HEK293T同時強迫表現FADS2及ELOVL2可以使該細胞株DHA合成效率顯著增加。 故本論文推論：在參與DHA合成的一系列酵素中，FADS2或ELOVL2缺乏是成人無法有效率合成DHA的主要原因。
並列摘要
Docosahexaenoic acid (DHA, 22:6n-3) is mainly enriched in the brain and is essential for normal neurological function. DHA is converted from its precursor α-linolenic acid (ALA, 18:3n-3) by a series of long chain fatty acid desaturases (FADS2 and FADS1) and elongases (ELOVL2 and ELOVL5). During brain development, infant has the ability to synthesize DHA, while in adult, DHA biosynthesis is low. We then studied whether some specific enzymes involved the DHA biosynthesis may be defected, and this defection can be overcome by its overexpression. The human cell lines, SH-SY5Y, MCF-7 and HEK 293T were incubated with DHA precursors, ALA, EPA (20:5n-3), DPA (22:5n-3), 24:5n-3 or 24:6n-3 to examine the potential defected enzymes to block DHA biosynthesis. The fatty acid composition was analyzed by gas chromatography with flame ionization dectector. The enzymes involved in DHA biosynthesis, FADS2, FADS1, ELOVL5 and ELOVL2 mRNA expression were analyzed by RT-qPCR. We found the mainly defected enzymes involved DHA biosynthesis was ELOVL2 in SH-SY5Y, FADS2 in MCF-7 or both FADS2 and ELOVL2 in HEK 293T. The DHA biosynthesis from its precursors were recovered by the overexpressed FADS2 or ELOVL2, or FADS2 plus ELOVL2 in MCF-7, SH-SY5Y or HEK 293T, respectively. It is suggested that FADS2 or/and ELOVL2 may be defected for the DHA biosynthesis in adults.
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