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研究生:陳佳妤
研究生(外文):Chia-Yu Chen
論文名稱:藉由多元不飽和脂肪酸調控粒線體功能提供Ceramide對神經膠細胞毒性之保護作用
論文名稱(外文):Functional Modulation of Mitochondria by Polyunsaturated Fatty Acids Provide Protection against Ceramide Toxicity to Glioma Cells
指導教授:謝榮鴻
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:保健營養學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:97
中文關鍵詞:多元不飽和脂肪酸神經保護
外文關鍵詞:neuroprotectionpolyunsaturated fatty acids
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現今已有研究指出,多元不飽和脂肪酸 (polyunsaturated fatty acids, PUFAs) 具有神經保護的功能性,並能延緩神經退化性疾病。亦有文獻顯示,脂肪酸可藉由影響轉錄因子調控基因表現,因此本研究旨在探討藉由ceramide誘導神經退化模式,並以二十碳四烯酸 (arachidonic acid;C20:4 n-6) 及二十碳五烯酸 (eicosapentaenoic acid;C20:5 n-3) 處理神經膠瘤細胞,對於粒線體生合成相關基因及其上游轉錄因子基因表現之影響,並觀察其保護作用。以C6神經膠瘤細胞作為研究對象,預處理或與ceramide同時處理二十碳四烯酸及二十碳五烯酸24小時,觀察細胞型態及檢測細胞存活率,並檢測粒線體功能性。以二十碳五烯酸預處理或與50μM ceramide同時處理24小時後,細胞型態及存活率與ceramide組相較皆明顯上升,且粒線體功能回復。在基因表現方面,觀察上游轉錄因子PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1 α)、Tfam (mitochondrial transcriptional factor A) 之轉錄活性皆顯著上升,進而測定粒線體基因之ND6 (NADH dehydrogenase subunit 6) mRNA表現亦有明顯增加之情形。然而二十碳四烯酸預處理或與ceramide同時處理24小時後,細胞型態及存活率與ceramide組相較雖明顯上升,但粒線體功能並無明顯回復。在轉錄因子活性方面,觀察Tfam之轉錄活性明顯上升,且粒線體基因ND6之mRNA表現亦有回復之情形。由上述實驗數據推論二十碳五烯酸為經由促進PGC-1α之表現,而二十碳四烯酸則並非完全經由PGC-1α途徑,達到刺激粒線體生合成之效果;並發現以二十碳五烯酸處理具有預防或回復ceramide所造成之粒線體功能性傷害,以達到神經細胞保護作用。
Mitochondrial dysfunction has been reported in the pathogenesis of neuron degeneration. Recent studies indicated that polyunsaturated fatty acids (PUFAs) have beneficial effects on various neuron functions and mitochondrial biogenesis. Besides, the effect of gene regulation by fatty acids is considered about their specific binding to nuclear receptors as transcription factors, or changed in abundance of these transcription factors indirectly. In the current study, we created a C2 ceramide-induced neurodegeneration model of C6 glioma cells line. Various concentrations of arachidonic acid (AA; n-6 C20:4) or eicosapentaenoic acid (EPA; n-3 C20:5) were treated to evaluate the effects of neuroprotection and its regulatory mechanism. The cell viabilities were decreased by ceramide treatment for 24 h. The cell viabilities of PUFAs pre-treatment 24 h followed by exposure to 50 μM ceramide were also determined. The C6 cells pre-treated with EPA showed significant resistance to ceramide cytoxicity. The cell viabilities were increased in the cohorts of EPA and AA treatment in both of PUFAs and ceramide incubated at the same time. Increased transcriptional activities of mitochondrial transcriptional factor A (Tfam) were examined by promoter activities assay from cells pre-treatment and co-treatment of EPA and AA. Furthermore, the PUFAs could induce mitochondrial mRNA expressions were observed in current study. These data represented that PUFAs could provide protective effects on cells from the attack of ceramide, and more correlation to the modulated mitochondrial function.
摘 要 I
Abstract III
目錄 IV
表目錄 X
圖目錄 XI
第一章 序論 1
第二章 文獻回顧 2
第一節 神經膠細胞與神經保護作用 2
第二節 粒線體與電子傳遞鏈 3
第三節 神經退化性疾病及粒線體功能不全之相關性 4
第四節 多元不飽和脂肪酸可能達到神經保護作用之機制 5
第五節 轉錄因子調控粒線體生合成作用 6
第六節 粒線體轉錄因子A (Mitochondrial transcriptional factor A) 7
第七節 過氧化小體增生活化接收器γ共同活化子-1α (Peroxisome proliferator-activated receptor γ coactivator 1 α) 8
第三章 研究目的 10
第四章 材料與方法 11
第一節 質體構築 11
(一) 建構Tfam Pro-Luc報導質體 11
(二) 建構PGC-1α Pro-Luc報導質體 11
第二節 勝任細胞 (competent cells) 之製備 12
第三節 轉型作用 (transformation) 12
第四節 質體DNA之萃取 13
第五節 細胞培養 14
(一) 細胞株 14
(二) 培養條件 14
第六節 藥劑處理 14
(一) 脂肪酸 14
(二) Ceramide 15
第七節 實驗組別 15
(一) 控制組 15
(二) 負控制組 15
(三) 正控制組 15
(四) Pre-treatment組 16
(五) Co-treatment組 16
第八節 細胞存活率測試 16
第九節 細胞之短暫轉染 (transient transfection) 17
第十節 冷光酶報導基因分析 17
第十一節 RNA萃取 18
第十二節 反轉錄作用 19
第十三節 同步定量反轉錄聚合酶連鎖反應 (Real-time reverse transcriptional polymerase chain reaction, real-time RT-PCR) 19
第十四節 細胞蛋白質之萃取 20
第十五節 蛋白質之定量 20
第十六節 西方點墨法分析 21
第十七節 流式細胞儀分析 22
第十八節 統計分析 23
第五章 實驗結果 24
第一節 對於C6 神經膠瘤細胞型態之影響 24
(ㄧ) Ceramide處理對於C6 神經膠瘤細胞型態之影響 24
(二) 多元不飽和脂肪酸對於C6 神經膠瘤細胞型態之影響 24
(三) Pre-treatment組對於C6 神經膠瘤細胞型態之影響 25
(四) Co-treatment組對於C6 神經膠瘤細胞型態之影響 25
第二節 對於C6 神經膠瘤細胞存活率之影響 26
(ㄧ) Ceramide處理對於C6 神經膠瘤細胞存活率之影響 26
(二) 多元不飽和脂肪酸對於C6 神經膠瘤細胞存活率之影響 26
(三) Pre-treatment組對於C6 神經膠瘤細胞存活率之影響 27
(四) Co-treatment組對於C6 神經膠瘤細胞細胞存活率之影響 28
第三節 觀察C6 神經膠瘤細胞粒線體膜電位之影響 30
(一) 給予ceramide對於C6 神經膠瘤細胞膜電位之影響 30
(二) 多元不飽和脂肪酸對於C6 神經膠瘤細胞粒線體膜電位之影響 30
(三) Pre-treatment組對於C6 神經膠瘤細胞粒線體膜電位之影響 30
(四) Co-treatment組對於C6 神經膠瘤細胞粒線體膜電位之影響 31
第四節 觀察C6 神經膠瘤細胞對粒線體相關轉錄因子基因表現之影響 32
(一) 投予多元不飽和脂肪酸對C6 神經膠瘤細胞轉錄因子基因表現影響 32
(二) Pre-treatment組對於C6 神經膠瘤細胞轉錄因子基因表現之影響 33
(三) Co-treatment組對於C6 神經膠瘤細胞轉錄因子基因表現之影響 34
第五節 觀察C6 神經膠瘤細胞對粒線體基因之影響 36
(一) Pre-treatment對C6 神經膠瘤細胞粒線體基因表現之影響 36
(二) Co-treatment對C6 神經膠瘤細胞粒線體基因表現之影響 37
第六章 討論 39
第一節 處理ceramide對於傷害C6 神經膠瘤細胞之影響 39
第二節 給予多元不飽和脂肪酸對於C6 神經膠瘤細胞之影響 40
第三節 多元不飽和脂肪酸處理對於C6 神經膠瘤細胞達到之保護作用 41
(一) Pre-treatment組對於C6 神經膠瘤細胞保護作用之影響 41
(二) Co-treatment組對於C6 神經膠瘤細胞保護作用之影響 42
第四節 多元不飽和脂肪酸對轉錄因子及相關粒線體基因表現之影響 43
(一) 觀察多元不飽和脂肪酸處理C6 神經膠瘤細胞對轉錄因子之影響 43
(二) 觀察Pre-treatment處理多元不飽和脂肪酸對C6 神經膠瘤細胞轉錄因子之影響 44
(三) 觀察Co-treatment處理多元不飽和脂肪酸對C6 神經膠瘤細胞轉錄因子之影響 45
第五節 綜合討論 47
(一) Ceramide影響C6 神經膠瘤細胞粒線體之基因表現造成傷害之作用 47
(二)多元不飽和脂肪酸調控轉錄因子之表現以達到神經細胞之保護作用 47
第七章 結論 49
第八章 參考文獻 50
第九章 附錄 56
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