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研究生: 張佳鼎
Chia-Ting Chang
論文名稱: 促進神經元分化之中草藥藥物篩選
Screening for neurogenic Chinese herbal medicines
指導教授: 王慈蔚
Wang, Tsu-Wei
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2014
畢業學年度: 102
語文別: 中文
論文頁數: 43
中文關鍵詞: 神經幹細胞成年神經新生中草藥Wnt/β-catenin訊息傳導路徑
英文關鍵詞: Neural stem cell, Adult neurogenesis, Chinese herbal medicines, Wnt/β-catenin pathway
論文種類: 學術論文
相關次數: 點閱:157下載:1
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  • 現今有許多的神經退行性疾病像是阿茲海默症和帕金森氏症都會導致神經不可逆轉的死亡,進而損害大腦的功能。而在大腦中存在的神經幹細胞則提供了治療可能性,透過誘導神經幹細胞分化成成熟的神經元,就有可能可以補充因神經退行性疾病所引起的神經損失。為了篩選有促進神經元分化之中草藥,我們首先使用P19細胞株來進行第一階段的篩選。P19是一類似多功能幹細胞的細胞株,隨著我們給予不同的藥物,或是轉染不同的神經性bHLH (basic helix-loop-helix) 轉錄因子,P19細胞可以分化成神經或是肌肉細胞。在第一輪篩選的過程中我們發現,在不同濃度的順天堂及工研院中草藥物處理下的P19細胞,約有17%的中草藥有促進神經元分化的效果。而在第二輪篩選,我們利用已知促進神經元分化的藥物維甲酸進行前懸浮培養,再來看通過第一輪篩藥的中草藥萃取物能否再更進一步的促進P19細胞神經元分化,最後約有5.1%中草藥通過了第二輪的篩選。為了更進一步研究其促進神經元分化的作用,我們將神經幹細胞從出生後7天小鼠大腦的腦室下區取出並培養形成神經球,之後以通過第二輪篩選的中草藥培養分化。我們發現NH003和NH005可促進神經幹細胞進行神經元分化。我們更進一步發現NH003和NH005會促進與神經元分化有關的Wnt訊息傳導路徑下游分子β-catenin的表現。為了探討 NH003和NH005是否透過增加β-catenin來促進神經元分化,我們轉染shβ-catenin後給予NH003和NH005來培養P19細胞,發現NH003和NH005促進神經元分化的效果被抑制,可以得知NH003和NH005促進神經元分化的效果可能是透過Wnt 訊息傳導路徑所達成的。為了確保NH003和NH005是專一促進神經元分化,而非促進細胞增生所引起的效應,我們在給予NH003和NH005培養下,於固定細胞的前兩個小時同時給予BrdU來標記正在增生中的細胞,我們發現與控制組相比NH003或NH005的組別細胞增生的數量並沒有明顯差異。這些結果顯示,NH003和NH005會透過Wnt 訊息傳導路徑來誘導神經元分化並且不影響細胞增生。而NH003中的銀杏內脂A可能是銀杏萃取物中引發神經元分化的成分之一。我們的研究發現中草藥中的銀杏具有誘導神經元分化的功能。

    Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease cause irreversible neuronal death that impairs brain functions. The discovery of neural stem cells in the adult brain provides possibilities that we can induce these cells to differentiate into neurons lost in neurodegenerative disorder. To screen for potential neurogenic compounds, we started with P19 cell cultures. P19 cells are pluripotent stem cell-like cells, which can be induced to differentiate into neural and muscle cells by drug treatments or transfected with neural basic helix-loop-helix (bHLH) transcription factors. In the first round of screening, P19 cells were cultured with various Chinese herbal extracts of different concentrations provided by Sun Ten Pharmaceutical or Industrial Technology Research Institute for 3 days. We found that 17% of screened Chinese herbal extracts induced neuronal differentiation in this culture condition. In the second round of screening, P19 cells were cultured to form aggregates with retinoic acid (RA), a known molecule to induce neuronal differentiation, first and then cultured as monolayer with those herbal extracts identified from the first round of screening. After the second round of screening, we found that 5.1% of screened Chinese herbal extracts, including NH002, NH003, NH005 (from Sun Ten Pharmaceutical) and drug 379 (from Industrial Technology Research Institute) promoted neuronal differentiation. To further confirm their neurogenic effect, neural stem cells isolated from the subventricular zone of the postnatal day 7 mouse brain were cultured to form neurospheres. Dissociated neurosphere cells were treated with Chinese herbal extracts from the second round of screening. We found that NH003 and NH005 promote neuronal differentiation in neural stem cells. Furthermore, we found that NH003 and NH005 increased the expression of β-catenin, the key effector protein of the Wnt signaling known for its role in neuronal differentiation. To examine whether β-catenin mediates the neurogenic effect of NH003 and NH005, P19 cells were transfected with shRNA against β-catenin and cultured with NH003 or NH005. We found that knockdown of β-catenin blocked neuronal differentiation in NH003 and NH005 culture. To make sure the neurogenic effect was not due to increased cell proliferation, we added BrdU two hours before fixation to label proliferating cells in NH003 or NH005 treatments. BrdU-positive cell numbers were not changed during NH003 or NH005 group. Therefore, NH003 and NH005 have no effect on cell proliferation. Taken together, our study suggests that NH003 and NH005 promote neuronal differentiation through Wnt/β-catenin pathway without affecting proliferation. Lastly, we found that ginkgolid A could be one of the effective components from NH003 (ginkgo biloba) to induce neuronal differentiation. In conclusion, Chinese herbal medicines could be potential treatments for neurodegenerative disorders.

    摘要………………….……………..………………...….…..…3 English abstract…………………………………..…….….……5 1. Introduction……….……………..………………...….…..…7 1.1 Neurodegenerative disorder 1.2 Stem cell therapy 1.3 Adult neurogenesis 1.4 Chinese herbal therapy 2. Materials and Methods………………………….…..…..….11 2.1 Chinese herb medicines 2.2 P19 cell culture and transfection 2.3 Primary cell cultures 2.4 Immunostaining analysis 2.5 Western blotting 2.6 Knockdown experiment 2.7 Image analysis and statistical analysis 3. Results………………..……………………………..…...…16 3.1 P19 cells can differentiate into neurons 3.2 First round of screening 3.3 Second round of screening (RA-induced) 3.4 CHMs induce neuronal differentiation of neural stem cells 3.5 NH003 and NH005 promote neuronal differentiation through the canonical Wnt signaling pathway 3.6 NH003 and NH005 have no effect on cell proliferation 3.7 Ginkgolid A could be one of the effective components in NH003 to promote neuronal differentiation 3.8 Ginkgolid A may not promote neuronal differentiation in NSCs 4. Discussion………………………………….….……...……22 4.1 Wnt pathway 4.2 ERK pathway 4.3 EGb761 4.4 Ginkgolides 5. Table……………………………………….….……...……26 6. Figure……………………………………….….…..………28 7. References……………………………….…...………….…39

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