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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 蔣丙煌(Been-Huang Chiang) | |
dc.contributor.author | Wen-Ting Hsieh | en |
dc.contributor.author | 謝雯婷 | zh_TW |
dc.date.accessioned | 2021-06-16T05:40:20Z | - |
dc.date.available | 2019-08-21 | |
dc.date.copyright | 2014-08-21 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-12 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56656 | - |
dc.description.abstract | 本研究的實驗目的是以人類胚幹細胞株(human embryonic stem cell, hESC)H9細胞株建立一體外平台,用以模擬中腦多巴胺神經細胞(midbrain dopaminergic neurons, mDA neurons)的發育過程,得以藉此篩選出具有刺激神經細胞新生潛力的植化素,並擬探討此最具有刺激神經細胞新生功效之活性成分的作用機制。結果顯示,確可透過一個五階段(Stage I-V)的分化流程,成功使H9細胞株分化形成類mDA的神經細胞,其形成TH+細胞的分化率為32.8-53.6%,並進一步透過mDA相關因子如En1、Lmx1a、TH(tyrosine hydroxylase)、AADC(aromatic L-amino acid decarboxylase)與GIRK2等之mRNA表現分析,以及相關的特定蛋白如nestin、TH、DAT(dopamine transporter)與β-III tubulin之免疫螢光染色分析,證明所分化的細胞確為類似mDA神經細胞。另外,透過與維持神經電生理相關的離子通道之mRNA表現分析與多巴胺分泌量的測定,確立了分化而成的細胞具有神經功能性,並發現此流程可使細胞產生具mDA特定性的Kv10.1(Kv: voltage gated-potassium channel)表現。在確立了分化流程後,我們更進一步利用過去文獻發現具有刺激神經新生的藥物多巴胺受器促進劑bromocriptine、7-OH-DPAT與抑制劑sulpiride驗證此平台的效果,其方法為分析兩DA神經細胞之特定蛋白TH與GIRK2的qPCR表現量,以此分別確立了上述正、負控藥物的有效濃度為:20 μM bromocriptine、10 μM 7-OH-DPAT與10 μM sulpiride。最後,在植化素成分的篩選上,發現綠茶兒茶素效果與其結構上的差異相關,而EGCG(epigallocatechin gallate)為其中最具效果的食品成分,其最佳處理濃度為12.5 μM;而人參皂苷Rb1的處理結果則顯著優於綠茶兒茶素,當其處理濃度為6.25 μM時,可大幅提升TH與GIRK2的表現量分別達12與14倍,顯示其在促成mDA神經細胞分化形成上的潛力。而在相關機制探討中發現,Rb1可能是透過促使周圍環境的星狀細胞分泌相關的神經滋養因子BDNF(brain derived neurotrophic factor)與NT-3(neurotrophin-3),以及誘使Wnt-1/ frizzled-1/β-catenin傳導途徑的產生,而具有促類似mDA神經細胞分化的能力。 | zh_TW |
dc.description.abstract | This study aimed to establish an in-vitro model using hESC (human embryonic stem cell) H9 cell line, which is expected to simulate the development of midbrain dopaminergic neurons (mDA neurons). Thus, through the model, we expected to screen phytochemicals with potential on stimulating mDA neurogenesis to improve Parkinson's disease (PD). As a result, we successfully established a five-stage process which could differentiate hESC H9 cell line into mDA-like neurons, and the differentiation rate of TH+ cells was 32.8-53.6%. Moreover, the final differentiated DA neurons truly expressed mature DA neuron-related factors like En1, Lmx1a, TH (tyrosine hydroxylase), AADC (aromatic L-amino acid decarboxylase), GIRK2 and other factors. Through the analysis of immunefluorescence staining, the in-vitro model also showed specific protein expression such as nestin, TH, β-III tubulin and DAT (dopamine transporter). These results demonstrated these differentiated cells were certainly mDA-like neurons. Besides, these cells showed mRNA expression of several voltage-gated ion channels, especially the mDA-specific one, Kv 10.1. In addition, the dopamine release (7.28 ± 1.30 ng/ml) by the differentiated cells was also detected. After that, according to the qPCR analysis of two marker genes, TH and GIRK2, we determined bromocriptine and 7-OH-DPAT as positive control drugs and sulpiride as negative control suitable for this model. Their effective dosages are presented as the following: 20 μM bromocriptine, 10 μM 7-OH-DPAT and 10 μM sulpiride. Finally, we tested green tea catechins (GTCs), including EGCG, ECG, EGC and EC as well as ginsenoside Rg1, Rg3 and Rb1 using the established model. We found that the abilities of GTCs might be related to their differences in structure. EGCG, which possesses both galloyl moiety and tri-hydroxyl substitutes on the B-ring, significantly increased the TH and GIRK2 expression at 12.5 μM. Moreover, better than GTCs, at 6.25 μM, ginsenoside Rb1 greatly enhanced the TH and GIRK2 expression to 12 and 14 folds, respectively. Thus, it appeared that Rb1 was the most potent phtyochemical to have potential of stimulating the differentiation of mDA-like neurons. Its possible mechanism might include stimulating the BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3) secretion from surrounding astrocytes and activting the Wnt-1/ frizzled-1/β-catenin signaling pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:40:20Z (GMT). No. of bitstreams: 1 ntu-103-F96641006-1.pdf: 4401406 bytes, checksum: 0f30281fcb1ed9124c50f22828adfc9c (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 摘要 I
ABSTRACT II 圖目錄 VI 表目錄 VIII 第一章、緒論 1 第二章、文獻整理 3 第一節、帕金森氏症(PARKINSON’S DISEASE)的相關介紹 3 2-1-1. 症狀與病理特徵(Symptoms and Pathology) 3 2-1-2. 成因(Etiology) 7 2-1-3. 治療方式 11 第二節、神經細胞新生(NEUROGENESIS)與幹細胞(STEM CELLS) 16 2-2-1. 成年神經細胞新生(Adult Neurogenesis) 16 2-2-2. 黑質體中之多巴胺神經細胞新生(Dopaminergic Neurogenesis in SN) 17 2-2-3. 具多巴胺神經細胞分化潛力的幹細胞 21 2-2-4. 影響人類胚幹細胞(hESC)分化形成多巴胺神經細胞的因子 24 第三節、評估食品原料常用的帕金森氏症模式 30 2-3-1. 常用來模擬帕金森氏症的神經毒物(Neurontoxin) 30 2-3-2. 細胞膜式 32 第四節、具抗帕金森氏症潛力的食品原料 34 2-4-1. 綠茶(Green Tea) 34 2-4-2. 人參(Ginseng) 35 第五節、食品原料影響體內神經細胞新生的可能途徑 37 第三章、實驗目的與實驗架構 40 第一節、實驗目的 40 第二節、實驗架構 41 3-2-1. 體外平台的建立與驗證 41 3-2-2. 潛力食品成分的篩選 42 第四章、實驗材料與方法 43 第一節、實驗材料 43 第二節、實驗儀器 45 第三節、實驗方法 46 4-3-1. 細胞培養─人胚幹細胞(Human Embryonic Stem Cells, hESCs)分化流程 46 4-3-2. 相關基因表現檢測 49 4-3-3. 細胞免疫螢光染色法(Immunofluorescence Staining, IF) 58 4-3-4. 以流式細胞儀測定細胞的分化比例 60 4-3-5. 以流式細胞儀測定細胞存活率 60 4-3-6. 鈣離子通道測定 60 4-3-7. 多巴胺(dopamine, DA)含量分析 61 4-3-8. 統計方法 62 第五章、實驗結果與討論 63 第一節、建立H9細胞株分化至類中腦多巴胺神經細胞(MDA-LIK3 NEURON)流程 63 5-1-1. 類mDA神經細胞分化流程的確立 63 5-1-2. 分化過程中細胞之分化率的結果 71 第二節、分化過程相關的細胞鑑定 74 5-2-1. 相關轉錄因子(Transcription Factors)與標定基因的mRNA表現情形 74 5-2-2. 細胞分化過程相關的蛋白質表現情形 80 5-2-3. 與維持神經電生理相關的離子通道表現情形 86 第三節、正、負控藥物的處理結果 93 5-3-1. 分化之多巴胺神經細胞上的多巴胺受器(DR)mRNA表現情形 93 5-3-2. TH與GIRK2之mRNA表現情形 97 第四節、綠茶多酚(GREEN TEA POLYPHENOLS, GTPS)的處理結果 103 第五節、人參皂苷(GINSENOSIDE)的處理結果 113 第六節、以NESTIN為指標測試具促神經新生的潛力化合物 125 第六章、結論 132 第七章、未來實驗方向 134 第一節、人參皂苷RB1作用機制的確立 134 第二節、其他潛力植化素的篩選 137 第三節、體內試驗的驗證 140 第八章、參考文獻 141 | |
dc.language.iso | zh-TW | |
dc.title | 利用人胚幹細胞建立一篩選具刺激多巴胺神經細胞分化潛力以改善帕金森氏症之植化素的體外模式 | zh_TW |
dc.title | An in-vitro model derived from hESC for screening phytochemicals with dopaminergic differentiation-boosting potential for improving Parkinson’s disease | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 錢宗良(Chung-Liang Chien),李宣書(Hsuan-Shu Lee),潘建源(Chien-Yuan Pan),吳信志(Shinn-Chih Wu) | |
dc.subject.keyword | 帕金森氏症,黑質體緻密部,A9型多巴胺神經細胞,人類胚胎幹細胞,綠茶兒茶素,人參皂?, | zh_TW |
dc.subject.keyword | Parkinson’s disease (PD),substantia nigra pars compacta (SNpc),type A9 dopaminergic neurons,human embryonic stem cells (hESCs),green tea catechins (GTCs),ginsenosides, | en |
dc.relation.page | 203 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-12 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 食品科技研究所 | zh_TW |
顯示於系所單位: | 食品科技研究所 |
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