臺灣博碩士論文加值系統

神經退化性疾病例如阿茲海默症為神經細胞發生漸進式退化與功能漸失，進而發生神經細胞死亡等現象所引起的疾病，影響腦部的神經退化性疾病常見病人大腦中神經細胞功能受到影響，並且伴隨認知與學習記憶的功能退化。已有許多文獻發現，蔬果富含的類黃酮化合物例如柑橘類黃酮可以通過血腦障壁，並具有類似神經滋養因子的活性，促進腦部神經新生、神經突出分枝生長、神經分化與神經存活等功能。本論文的主要目的為探討柑橘多甲基氧類黃酮5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF)是否具有促進神經細胞突出分枝生長(neurite outgrowth)與分化(differentiation)等類似神經滋養因子的功能，並且研究其神經滋養功效上參與的分子訊息路徑。
於本論文的研究結果中，我們發現柑橘多甲基氧類黃酮化合物5-OH-HxMF具有誘導PC12細胞突出分枝生長的功能。接著我們利用即時定量聚合酶鏈鎖反應以及西方墨點法分析發現，5-OH-HxMF誘導PC12細胞突出分枝生長時，伴隨著神經細胞分化標記基因growth associated protein-43 (GAP-43) mRNA與蛋白質的表達，此結果證明5-OH-HxMF具有促進神經細胞分化的能力。接下來我們進一步研究5-OH-HxMF促進神經細胞分化的分子訊息路徑。首先，我們針對調控神經分化相關的cAMP response element binding protein (CREB)蛋白質進行分析，我們利用西方墨點法分析CREB磷酸化的情形，結果發現在PC12細胞中5-OH-HxMF會增加磷酸化CREB的量，並且經由抑制劑抑制磷酸化CREB活性的實驗結果證明，5-OH-HxMF活化的CREB與CREB binding protein(CBP)形成複合體，進一步與CRE DNA序列結合，進而活化CRE下游所調控基因的轉錄作用。
接下來，我們利用可能影響CREB磷酸化的分子訊息路徑抑制劑，包括U0126、 Bisindolylmaleimide I、LY294002 、KN62、SQ22536與 H-89等，探討5-OH-HxMF可能經由哪一條分子訊息路徑活化CREB。我們的實驗結果發現，5-OH-HxMF 主要是藉由影響cAMP與protein kinase A (PKA)訊息路徑活化CREB，並促進CRE調控下游基因的轉錄作用，進而促進PC12細胞突出分枝生長與分化，然而我們也發現5-OH-HxMF活化CREB的活性可能與ERK/MAPK、PI3-K、PKC與CaMKII等分子訊息路徑無關。此外，我們進一步發現5-OH-HxMF快速增加PC12細胞內cAMP的量以及PKA之活性，最後我們抑制5-OH-HxMF在PC12細胞內誘導產生的cAMP以及PKA活性，發現也抑制了5-OH-HxMF 所誘導PC12細胞突出分枝生長的現象，此結果證明5-OH-HxMF經由cAMP/PKA分子訊息路徑活化CREB，進而影響PC12細胞突出分枝生長的現象。
總結本論文的實驗結果，5-OH-HxMF 具有類似神經滋養因子誘導神經分化的功能，其分子機轉為5-OH-HxMF藉由增加細胞內cAMP的量，進而活化PKA活性，進一步促進CREB磷酸化，活化的CREB與CBP結合後，進一步結合具有CRE DNA序列的基因，並促進CRE調控下游與神經分化相關基因表達，促使PC12細胞突出分枝生長與神經分化。本研究結果將可以提供柑橘類黃酮化合物未來應用於神經退化性疾病預防與治療藥物開發的基礎。

Neurodegenerative diseases such as Alzheimer’s disease (AD) are characterized by the progressive degeneration, dysfunction and loss of neuronal cells in the nerve system. Patients with neuronal dysfunction in the brain accompanied losses of cognition and learning memory. Recently, several evidences suggest that the neurotrophic action of dietary flavonoids, such as citrus flavonoids, may transverse the blood-brain-barrier (BBB) and exert beneficial effects on neurogenesis, neuronal differentiation, neuronal survival, and may prevent cognitive losses associated with neurodegeneration. The aim of this study is to investigate the neurotrophic effects and mechanism of 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a citrus hydroxyl polymethoxyflavone (PMF), on promoting neuronal cell neurite outgrowth and differentiation.
In the present study, we found that 5-OH-HxMF significantly induces neurite outgrowth along with increased mRNA and protein expression of the neuronal cell differentiation marker, growth-associated protein-43 (GAP-43), in PC12 cells. These results indicated that 5-OH-HxMF could promote neuronal cell differentiation. It is known that activation of cAMP response element binding protein (CREB) may play an essential role in neuronal cell differentiation, we investigated whether 5-OH-HxMF activate CREB in PC12 cells. We found that 5-OH-HxMF stimulated phosphorylation of CREB protein and activated the transcription of cAMP response element (CRE)-mediated luciferase reporter gene, which was inhibited by KG-501, a specific antagonist for the CREB-CBP complex. To identify the molecular signaling pathways involved in the CREB activation, the pathway selectively specific inhibitors including U0126, Bisindolylmaleimide I, LY294002, KN62, SQ22536 and H-89 were used. Our results show that 5-OH-HxMF induced CREB activation significantly attenuated by adenylate cyclase inhibitor (SQ22536) and protein kinase A (PKA) inhibitor (H-89), respectively. Moreover, we also found that CREB activation is independent on the ERK / MAPK, PI3-K, PKC and CaMK II signaling pathways. Our results indicated that 5-OH-HxMF stimulated CREB phosphorylation through the cAMP/PKA pathway. Consistently, our results show that 5-OH-HxMF increased the level of intracellular cAMP rapidly and downstream component, PKA activity in PC12 cells. Finally, we found that the adenylate cyclase inhibitor SQ22536 and the PKA inhibitor H-89 significantly blocked the potentiation of 5-OH-HxMF-induced neurite outgrowth.
In conclusion, the above findings reveal that 5-OH-HxMF possessed the neurotrophic action for promoting neurite outgrowth and neuronal cell differentiation. 5-OH-HxMF induced neurite outgrowth via increases of intracellular cAMP levels and PKA activity, further enhanced CREB phosphorylation and CRE dependent transcription in PC12 cells. This study thus enhances our understanding on 5-OH-HxMF may be beneficial for prevention and therapeutic use for neurodegenerative disorders.

中文摘要 I
英文摘要 III
緒論 1
1.神經退化性疾病(neurodegenerative disorders)的重要性 1
2.神經滋養因子(neurotrophic factors)與神經退化性疾病 1
3.Cyclic-AMP response element binding protein (CREB)蛋白質分子影響神經細胞之功能 2
4. 影響CREB磷酸化之分子訊息路徑 3
5.具有類似神經滋養與促進神經細胞功能之類黃酮化合物 (flavonoids)研究 5
6.柑橘類黃酮(Citrus flavonoids)之研究 6
7.研究動機與目的 7
材料與方法 9
1.試劑與藥品 9
2.細胞培養(Cell culture) 11
3.細胞存活率測試(MTT assay) 11
4.Neurite outgrowth的分析 12
5.抑制劑的使用 13
6.神經細胞分化標記(marker)基因的分析 15
7.西方墨點法(Western blotting) 16
8.Reporter assay 18
9.cAMP量的測定 19
10.PKA avtivity測定 21
11.統計分析 23
結果 24
1.柑橘類黃酮化合物5-OH-HxMF與nobiletin對PC12細胞毒性之影響 24
2.柑橘類黃酮化合物5-OH-HxMF與nobiletin對PC12細胞 neurite outgrowth之影響 24
3.柑橘類黃酮化合物5-OH-HxMF對PC12細胞神經分化標記基因(neuronal differentiation marker) mRNA及Protein表達之影響 25
4.柑橘類黃酮化合物5-OH-HxMF與nobiletin對CREB磷酸化的影響 26
5.柑橘類黃酮化合物5-OH-HxMF對cyclic-AMP response element (CRE) 調控轉錄作用(transcription)之影響 27
6.柑橘類黃酮化合物活化CREB對PC12細胞neurite outgrowth 之影響 28
7.影響5-OH-HxMF 活化CREB之分子訊息路徑 29
8.柑橘類黃酮化合物5-OH-HxMF對細胞內cAMP 量及PKA活性之影響 30
9.柑橘類黃酮化合物5-OH-HxMF活化cAMP與PKA路徑對PC12細胞neurite outgrowth之影響 30
討論 32
參考文獻 54

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