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研究生：	廖彥婷 Liao, Yen-Ting
論文名稱：	探討NeuroD 轉錄因子對脂肪幹細胞神經分化與沒食子酸鹽衍生物對中樞神經細胞的影響 Studies on neural differentiation of adipose stem cells by NeuroD transcription factor and the effect of gallate derivates on CNS neural cells
指導教授：	曾淑芬 Tzeng, Shun-Fen
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	109
中文關鍵詞：	沒食子酸鹽、神經分化、中樞神經系統、脂肪幹細胞、轉錄因子
外文關鍵詞：	neuronal differentiation, CNS, transcription factor, gallate, adipose stem cells
相關次數：	點閱：185 下載：1
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第一部分: 探討NeuroD轉錄因子對脂肪幹細胞神經分化之影響
　　人類脂肪幹細胞（human processed lipoaspirate cells,簡稱hPLAs)爲近年來被發現具有幹細胞特性的一群細胞源，根據研究指出其具有發育骨源性（osteogenic）、脂肪源性（adipogenic）、肌源性（myogenic）、軟骨源性（chondrogenic）與神經源性（neurogenic）的潛力。NeuroD為一轉錄因子（transcription factor）；研究指出，NeuroD在神經發生（neurogenesis）的過程中扮演重要調節者的角色；且NeuroD的表現可作為幹細胞走向神經發生的重要指標。因此我們欲研究這些具有神經特化決定性的基因（neuronal specific determination genes），例如NeuroD，是否能透過基因調控，進而促進hPLAs神經分化。本研究中，我們利用b-mercapoethanol 誘導hPLAs神經發生，使其產生神經觸支（neurites）。此外，藉由免疫螢光染色法，發現到那些利用載體pcDNA將大鼠NeuroD基因轉殖的hPLAs，其細胞核有NeuroD蛋白表現，且細胞外觀亦有神經觸枝的表現。我們藉由大鼠神經膠質瘤細胞株（rat glioma cell line, C6），以觀測那些受到NeuroD基因轉殖所調控的下游分子（downstream molecules）的表現。結果顯示，轉殖pcDNA-NeuroD的C6有表現NeuroD所調控的神經標幟分子-神經纖維蛋白（neurofilament-145, NF-145與 neurofliament-200, NF-200）；推測NeuroD具有誘導C6細胞走向神經發生路徑的能力。在pcDNA-NeuroD基因轉殖的C6細胞與hPLAs外觀型態改變中，顯示過量表現NeuroD可促使hPLAs分化而形成神經細胞。

第二部分: 探討沒食子酸鹽衍生物對中樞神經細胞的影響
　　小膠質細胞（microglia）在中樞神經系統中扮演巨嗜細胞（macrophage）的角色；在中樞神經系統受傷後可被活化，並分泌前發炎調控因子（pro-inflammatory mediators），而造成中樞神經系統組織的二級傷害。因此，在中樞神經系統受傷後，抑制小膠質細胞活化可能對神經細胞存活有所助益，進而達到減緩中樞神經系統的傷害。

　　在植物與水果中的酚化合物甲基三羥基苯甲酯（methyl gallate, MG）與沒食子酸（gallic acid, GA），扮演著抗氧化物（antioxidants）、前誘導氧化物（pro-oxidants）、抗發炎因子（anti-inflammatory factors）與抗細菌（antibacterial）的角色，並具有清除游離自由基的潛力。我們發現在初級小膠質細胞、星狀細胞、大腦皮層神經細胞與C6、PC12細胞株，處理商業購得的MG後皆可促使細胞存活，但卻降低BV2（由小鼠小膠質細胞利用反轉錄病毒感染v-raf/v-myc致癌基因所產生的細胞株）的細胞存活。GA亦對BV2與星狀細胞具有細胞毒性的效果；在不管有無共同處理lipopolysaccharide (LPS)的大鼠初級小膠質細胞，GA都促進其細胞存活的作用。我們發現這二酚化物，尤其是MG，可能對持續增生的膠質細胞具有細胞毒害的作用。我們藉由觀測raf/myc細胞增生訊息傳遞路徑中MAP kinase磷酸化的程度；結果顯示，MG對BV2細胞的毒害機制，並非經由影響raf/myc細胞增生訊息傳遞路徑而導致。此外，我們亦探討MG對BV2細胞的毒殺作用是否起因於改變培養環境中鈉離子的濃度；其結果顯示，MG對BV2的毒殺作用機制並非導因於影響鈉離子通透的不平衡。我們亦藉由測量ATP生產量，以探討MG對BV2毒害作用是否造成粒線體功能缺損所導致；結果顯示，處理MG對BV2細胞ATP生產量具有劑量相依性的下降；推測MG對BV2細胞毒殺之機制，可能是造成細胞粒腺體功能損傷，而使細胞ATP產量受到影響，最後導致BV2細胞能量供應不足而走向細胞死亡。

Part I :Study on neural differentiation of adipose stem cells by NeuroD transcription factor
　Human processed lipoaspirate cells (hPLAs) have been recently characterized, and known to generate the osteogenic, adipogenic, myogenic, chondrogenic, and neurogenic lineages. NeuroD is a transcription factor, and many researches indicated that NeuroD plays an important mediator in the neurogenesis linkage. The expression of NeuroD could be a marker of stem cells that are processed toward neurogenesis linkage. We are particularly interested in study whether genetical modification with neuronal specific determination gene, such as NeuroD, can improve the neuronal differentiation of hPLAs. In this study, hPLAs cultured in the medium containing b-mercapoethanol had the extended neurites. Furthermore, by immunofluorescence, NeuroD was observed in the nucleus of hPLAs that were transfected with pcDNA3.1 vector carrying rat NeuroD gene (pcDNA-NeuroD). To examine the expression of downstream molecule(s) regulated by transfected NeuroD, we used C6 rat glioma cell line. Transfected C6 cells expressed neurofilament-145（NF-145）and neurofilament-200 (NF-200), suggesting that NeuroD mediates the differentiation of C6 cells toward neuronal lineage. The morphological examination of hPLAs and C6 with NeuroD transfection indicated that overexpression of NeuroD may force hPLAs to differentiate into neurons.

Part II : Study on the effect of gallate derivates on CNS neural cells
　Microglia, CNS resident macrophages, can be activated after CNS injury to produce cytotoxins such as pro-inflammatory mediators and induce secondary tissue damages that cause the progression of CNS diseases. Thus, deactivation of microglia after CNS injury can be beneficial for neuronal survival, leading to the reduction of CNS damage. Methyl gallate （MG）and gallic acid （GA）, phenolic compounds in plants and fruits, can act as antioxdiants, pro-oxidants, anti-inflammatory factors, antibacterial compounds, and have a potent free radical scavenging. We found that treatment with commercially available MG increased the cell viability of rat primary microglia, astrocytes, neurons, C6 cells and PC-12 cells, but reduced that of BV2 cells, a mouse microglial cell line generated by infecting with a v-raf/v-myc oncogene carrying retrovirus. GA also had cytotoxic effect on BV2 cells and astrocytes, while the compound increased the cell viability of rat primary microglia with or without bacterial lipopolysaccaride (LPS). Our findings suggest that the two compounds, especially MG, may have cytotoxic effect on proliferating glial cells. We investigated the effect of MG on the phosphorylation of MAP kinase of the raf/myc pathway in BV2 cells. The result showed that the cytotoxic effects of MG wasn’t by affecting this proliferating pathway. Furthermore, we investigated that if MG changed the Na+ gradient of BV2 cells. The time course of the assay showed that the cytotoxicity of MG in BV2 was not resulted from transient disruption of Na+ homeostasis. We also observed the ATP levels of the MG-treated BV2 cells, the result showed that the MG does-dependently decreased the ATP levels, indicating that the cytotoxic effects of MG on proliferating glial cells might cause by decreasing ATP levels of BV2 cells.

目錄………………………………………………………………………1
圖目錄……………………………………………………………………6
附圖目錄…………………………………………………………………8 
縮寫表.....................................................9

第一部分 : 探討NeuroD轉錄因子對脂肪幹細胞神經分化之影響……10

英文摘要…………………………………………………………………11
中文摘要…………………………………………………………………12
前言………………………………………………………………………14
一、幹細胞( stem cells)………………………………………………14
二、幹細胞的重要性…………………………………………………16
三、人體脂肪幹細胞（human processed lipoaspirate cells -hPLAs)..17
四、NeuroD與其重要性………………………………………………19
材料與方法..............................................21
一、材料………………………………………………………………21
（一）細胞培養材料…………………………………………………21
（二）化學藥品………………………………………………………21
（三）試劑組…………………………………………………………22
 (四）抗體……………………………………………………………23
  二、方法………………………………………………………………24
  （一）細胞培養………………………………………………………24
    1.初級人類脂肪幹細胞 --- hPLA cells……………………………24
    2.大白鼠神經膠質瘤細胞株--- C6 cell line………………………25
  （二）質體（plasmid）DNA的製備…………………………………25
  （三）基因轉殖（Transfection）……………………………………26
  （四）細胞免疫螢光染色（Immuno-fluorescence staining）………27
  （五）蛋白質萃取與西方點墨轉印法………………………………27
    1. 萃取細胞全部蛋白質……………………………………………27
    2.西方點墨轉印法（Western blotting）…………………………28
  （六）細胞存活分析法— MTT assay ………………………………29
  （七）細胞毒性分析法— LDH assay ………………………………29
結果…………………………………………………………………30
一、質體DNA的備製………………………………………………30
二、pcDNA-NeuroD基因轉殖在C6細胞株的表現…………………30
三、探討pcDNA-NeuroD基因轉殖在不同血清濃度下的表現……31
四、pcDNA-NeuroD基因轉殖對C6細胞株細胞型態的影響………32 
五、pcDNA-NeuroD基因轉殖誘導C6細胞株走向神經分化…………32
六、神經誘導初級人類脂肪幹細胞(hPLA cells)………………………33
七、pcDNA-NeuroD基因轉殖在hPLA細胞的表現…………………33
討論………………………………………………………………………35
一、NeuroD在hPLAs的作用………………………………………………35
二、NeuroD與其他bHLH family成員…………………………………38
三、未來研究方向與展望……………………………………………40
參考文獻…………………………………………………………………42	

第二部分:探討沒食子酸鹽衍生物對中樞神經細胞的影響……………………………62
英文摘要…………………………………………………………………63
中文摘要…………………………………………………………………65
前言......................................................67
一、小膠質細胞 (microglia)…………………………………………67
二、大鼠嗜鉻瘤細胞系細胞--- PC12…………………………………68
三、酚化合物(phenolic compounds)---甲基三羥基苯甲酯與沒食子酸....69
材料與方法………………………………………………………………72
一、材料………………………………………………………………72
（一）細胞培養材料…………………………………………………72
（二）化學藥品………………………………………………………72
（三）試劑組…………………………………………………………73
 (四）抗體……………………………………………………………74
二、方法………………………………………………………………74
（一）細胞培養………………………………………………………74
  1.初級小膠質細胞（primary microglia）…………………………75
  2.老鼠大腦皮層神經細胞 （primary brain cortical neurons）……75
  3.大白鼠神經膠質瘤細胞株（C6 cell line）………………………77
  4.小鼠小膠質細胞株（BV2 cell line）…………………………… 77
  5.大鼠嗜鉻瘤細胞系細胞（PC12）……………………………….78
（二）蛋白質萃取與西方點墨轉印法………………………………….78
    1. 萃取細胞全部蛋白質……………………………………………78
    2.西方點墨轉印法（Western blotting）……………………………78
（三）細胞存活（cell viability）分析法—MTT assay…………………79
（四）細胞毒性(cell cytotoxicity)分析法—LDH assay…………………80
（五）TNF-α、IL-1β與IFN-γ濃度分析……………………………80
（六）腺嘌呤核咁三磷酸(ATP)濃度分析………………………………80
結果………………………………………………………………………82
一、沒食子酸(gallic aicd , GA)對中樞神經系統細胞的影響…………82
二、甲基三羥基苯甲酯（methyl gallate, MG）對中樞神經系統細
胞的影響………………………………………………………………82
三、甲基三羥基苯甲酯（methyl gallate, MG）對初級小膠質細
胞的影響………………………………………………………………84
四、甲基三羥基苯甲酯（methyl gallate, MG）對BV2細胞毒殺
作用機制探討…………………………………………………………84
（一）探討mthyl gallat是否影響BV2細胞增生調控路徑…………84
（二）探討methyl gallate是否改變BV2細胞離子通透平衡………85
（三）探討探討methyl gallate是否影響BV2細胞ATP 產量…………86
討  論……………………………………………………………………88
一、Gallic acid與methyl gallte對中樞神經細胞的影響…………88
二、探討methyl gallte對BV2細胞毒殺機制…………………………89
三、未來研究方向與展望………………………………………………91
參考文獻..................................................93

                                    

第一部分: 探討NeuroD轉錄因子對脂肪幹細胞神經分化之影響
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第二部分: 探討沒食子酸鹽衍生物對中樞神經細胞的影響
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