臺灣博碩士論文加值系統

非亞甲基區隔脂肪酸 (non-methylene-interrupted fatty acids; NMIFA) 是一群存在於裸子植物種子內的罕見多元不飽和脂肪酸 (polyunsaturated fatty acid; PUFA)。由於NMIFA的價格昂貴且不易取得，以至於目前缺乏NMIFA代謝或生物活性的相關研究。先前的實驗結果顯示NMIFA具有抗發炎的功效，因此我們假設經由神經膠細胞攝取的NMIFA也能抑制神經發炎反應與調節生化介質的釋放。有鑑於此，我們先從竹柏種子中萃取sciadonic acid (SCA) 或以化學反應合成D7-eicosatrienoic acid (D7-ETrA)，再以矽膠管柱層析來分離與純化NMIFA；之後，利用脂多醣 (lipopolysaccharide; LPS) 刺激小鼠微膠細胞 BV-2 株的模型，來檢視pinolenic acid (PNA)、D7-ETrA 與SCA 可否經由代謝影響細胞內生化反應並抑制神經發炎反應。研究結果指出，PNA 可經由碳鏈加長作用轉變為 D7-ETrA；同時，在管柱層析的分離後，新合成的 D7-ETrA 與萃取出的 SCA可達98% 的高純度。當微膠細胞培養於添加PNA、D7-ETrA或 SCA的培養基，這三種NMIFA明顯地被細胞攝入，其中以 D7-ETrA最為顯著；同時NMIFA均能改變磷脂質脂肪酸組成並減少花生四烯酸 (arachidonic acid; AA) 的比例。在抗發炎活性的測試上，隨著濃度的增加，NMIFA皆能顯著地抑制一氧化氮 (nitric oxide; NO)、前列腺素 (prostaglandin E2; PGE2)、介白素-6 (interleuikin; IL-6) 與腫瘤壞死因子-a(tumor necrosis factor-a; TNF-a) 的生成；NMIFA在誘發性一氧化碳生成酶 (inducible nitric oxide synthase; iNOS)、第二型環氧酶 (type-2 cyclooxygenase; COX-2)、IL-6及TNF-a 的蛋白質或基因表現上也有顯著的抑制效果。進一步探討NMIFA對發炎訊息路徑mitogen-activated protein kinases (MAPK) 的可能影響，我們發現NMIFA能抑制 c-Jun N-terminal kinases (JNK) 訊息路徑，但對p38 MAPK (p38) 與extracellular signal-regulated kinases (ERK) 路徑卻有增加的趨勢，這兩種訊息的增加可能與提高神經保護因子及抗氧化酵素有關。總而言之， 三種NMIFA皆能被微膠細胞攝入而改變細胞磷脂質的脂肪酸組成，並可能藉由改變 MAPK 細胞訊
息傳導而調控發炎介質生成的基因表現。

Non-methylene-interrupted fatty acids (NMIFA) is a group of rare polyunsaturated fatty acid (PUFA) mainly found in gymnosperms seeds. Howver, D7-ETrA is not commercial available due to high production/ purification cost and limited sources, and few studies have been conducted to explore possible biological effect to date. Since NMIFA had previously been reported to exert anti-inflammatory effects, we hypothesized that NMIFA taken up by neuroglial cells might also inhibit neuronal inflammation and modulate the release of biochemical mediators. In this study, we extracted sciadonic acid (SCA) from podocarpus nagi seeds or synthesized D7-eicosatrienoic acid (D7-ETrA) from pine seeds oils, then separated and purified different NMIFA using column chromatography. Making use of murine microglia BV-2 cells as the inflammation model, we determined whether pinolenic acid (PNA), D7-ETrA or SCA could affect cellular biochemical reactions and relieve inflammatory responses induced by lipopolysaccharide (LPS). Results showed that SCA and D7-ETrA was separated and purified, and the purities of both fatty acids were approximate 98%. When microglia cells were incubated with PNA, D7-ETrA or SCA, all three NMIFA were incorporated into cellular phospholipids. The incorporation of NMIFA changed phospholipid fatty acid compositions, and decreased the proportion of arachidonic acid (AA). Increasing concentrations of PNA, D7-ETrA or SCA reduced the synthesis of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-6 (IL-6) and tumor necrotic factor-a (TNF-a) production. The suppression of pro-inflammatory mediators was due, in part, to the modified gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6 and TNF-a. The incorporation of NMIFA also suppressed phosphor-c-Jun N-terminal kinase (JNK), one of mitogen-activated protein kinases (MAPK), but enhanced phospho-extracellular signal-regulated kinase (ERK) and phosphor-p38 kinase. The increasing expression of both signaling proteins might be associated with the higher expression of neourotrophic factors and antioxidative enzymes. These findings indicate that NMIFA changes the phospholipid fatty acid composition, and modulates the responsiveness of microglia cells to LPS through modification of MAPK pathways.

目 錄 ......................................................................................................... VII
圖 目 錄 ................................................................................................... XII
表 目 錄 ................................................................................................... XV
第一章 前 言 ............................................................................................ 1
1.1 脂肪酸 .......................................................................................... 1
1.1.1 不飽和脂肪酸 ................................................................... 2
1.2 非亞甲基間隔脂肪酸 .................................................................. 5
1.2.1 Pinolenic acid (PNA) ........................................................... 6
1.2.2 D7-Eicosatrienoic acid (D7-ETrA) ....................................... 9
1.2.3 Sciadonic acid (SCA) ......................................................... 11
1.3 神經膠細胞 .................................................................................. 13
1.3.1 微膠細胞 (Microglia cell) ............................................... 14
1.3.2 Microglia cell激活 ............................................................ 16
1.4 免疫系統 ...................................................................................... 17
1.4.1 發炎反應 ........................................................................... 17
1.4.2 活性氧/氮分子 .................................................................. 18
1.4.3 細胞激素 ........................................................................... 21
1.4.4 脂質衍生物 ....................................................................... 24
1.4.5 發炎路徑 ........................................................................... 27
1.5 研究目的 ...................................................................................... 30
第二章 材料與方法 ................................................................................ 31
2.1研究架構 ....................................................................................... 31
2.2 實驗樣品 ...................................................................................... 31
2.3 實驗細胞株 .................................................................................. 31
2.4 實驗藥品與試劑 .......................................................................... 32
2.4.1 藥品與溶劑 ....................................................................... 32
2.4.2 免疫套組 ........................................................................... 33
2.4.3 實驗儀器與設備 ............................................................... 34
2.5 脂肪酸製備 .................................................................................. 35
2.5.1 D7-ETrA合成製備 ............................................................ 35
2.5.2 SCA製備 ........................................................................... 36
2.6 細胞培養 ...................................................................................... 36
2.6.1 細胞培養條件 ................................................................... 36
2.6.2 細胞活化 ......................................................................... 36
2.6.3 細胞繼代 ......................................................................... 36
2.6.4 細胞計數 ........................................................................... 37
2.6.5 細胞凍存 ........................................................................... 37
2.7 探討脂肪酸對LPS誘發微膠細胞後發炎物質的調控 ............. 38
2.7.1 細胞存活率測試 (MTT assay) ........................................ 38
2.7.2 一氧化氮測定 ................................................................... 40
2.7.3 Interleukin-6 (IL-6) 測定 .................................................. 41
2.7.4 Tumor necrosis factor-a(TNF-a) 測定 ............................ 42
2.7.5 前列腺素 Prostoglandin E2 (PGE2) 測定 ....................... 43
2.7.6 Western blot ........................................................................ 44
2.7.7 即時聚合酶連鎖反應 (Real-Time PCR) ........................ 47
2.8探討BV-2攝取脂肪酸後對脂質代謝的影響 ............................ 49
2.8.1細胞脂質萃取 .................................................................... 49
2.8.2脂肪酸分析 ........................................................................ 50
2.9 統計分析 ...................................................................................... 51
第三章 結果與討論 ................................................................................ 52
3.1D-ETrA合成 ............................................................................... 52
3.2探討NMIFA對BV-2細胞存活率之影響 ................................. 57
3.2.1細胞存活率測試 MTT assay ............................................ 57
3.3 NMIFA對BV-2細胞中脂肪酸組成與代謝的影響 .................. 59
3.3.1 攝取NMIFA 24小時後對BV-2細胞脂肪酸組成的影響..................................................................................................... 59
3.3.2 BV-2細胞內攝入NMIFA的總量與其分佈 ................... 60
3.3.3攝取NMIFA 24小時後對BV-2細胞磷脂質組成的影響 ..................................................................................................... 62
3.4以LPS誘導BV-2細胞來探討NMIFA之抗發炎活性 ............ 65
3.4.1探討NMIFA對BV-2細胞一氧化氮生成的影響 .......... 65
3.4.2探討NMIFA對BV-2細胞分泌細胞激素(IL-6)的影響 69
3.4.3探討NMIFA對BV-2細胞分泌細胞激素(TNF-a)的影響 ..................................................................................................... 70
3.4.4探討NMIFA對BV-2細胞分泌前列腺素E2之影響 ..... 72
3.5 NMIFA對BV-2細胞之蛋白質表現的影響 .............................. 74
3.5.1測定BV-2細胞中誘導型一氧化氮合成酶(iNOS)的表現 ..................................................................................................... 74
3.5.2測定BV-2細胞中第二型環氧酵素(COX-2)的表現 ...... 76
3.6測定NMIFA對BV-2細胞中mRNA的表現 ............................ 78
3.7探討NMIFA對BV-2細胞的發炎路徑 - MAPK的影響 ........ 83
第四章 結論 ............................................................................................ 88
第五章 參考文獻 .................................................................................... 91
第六章 附錄 .......................................................................................... 100
附錄一 ............................................................................................... 100
附錄二 ............................................................................................... 105
附錄三 ............................................................................................... 109
附錄四 ............................................................................................... 111
附錄五 ............................................................................................... 113

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