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研究生:林庭鋒
研究生(外文):LIN,TING-FENG
論文名稱:神經再程式化因子對人類神經幹/前趨細胞分化與人類尿液細胞神經再程式化之作用
論文名稱(外文):Effects of Neuronal Reprogramming Factors on the Differentiation of Human Neural Stem/Progenitor Cells and Neuronal Reprogramming of Human Urine cells
指導教授:許益超許益超引用關係
指導教授(外文):HSU,YI-CHAO
口試委員:鄒瑞煌李燕晉
口試委員(外文):CHOU,RUEY-HWANGLEE,YANN-JINN
口試日期:2017-01-24
學位類別:碩士
校院名稱:馬偕醫學院
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:45
中文關鍵詞:神經再程式化細胞分化人類尿液細胞
外文關鍵詞:UrineNeuronal Reprogramming FactorsNeural Stem/Progenitor CellsDifferentiationHuman Urine cells
相關次數:
  • 被引用被引用:0
  • 點閱點閱:264
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  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:0
人類神經細胞富含於大腦中,但是很難被取得。近期研究指出,人類神經細胞可以從誘導多功能幹細胞(iPSC)的分化或直接誘導神經元(iNs)從皮膚纖維母細胞的轉分化獲得。iPSC或iNs的來源也已經證明可以從皮膚纖維母細胞,血球細胞或肝細胞和尿液細胞形成。本研究目的是比較SH2B1(S)、miRNA124(I)、BRN2(B)、MYT1L(M)、NeuroD1(N)、ASCL1(A)、ATOH1(AT)等神經細胞特性因子對人類誘導的多功能幹細胞(iPSC)衍生的神經幹/前趨細胞(NSPC)中神經分化效率和活性氧化物產生的影響。此外,我們目的是選擇這些神經特性因子有用組合的方式,研究是否能藉由非侵入性的取得尿液細胞,並將其重編程為神經細胞。
實驗結果發現,經由慢病毒轉染上述神經細胞特性基因,都可觀察到這些基因在促進神經細胞及星狀分化有不同的效果,其中NeuroD1可產生最大量的神經細胞及最少量星狀細胞。每個基因促進神經分化後均造成ROS的微幅上升。在尿液細胞之神經再程式化方面,我們利用S-IBM重編程人類成纖維母細胞成TuJ1和MAP2(+)神經元細胞。更進一步地,我們證明只有IBMN可以直接重新編程尿液細胞形成具有神經元型態的TuJ1(+)細胞,S-IBM和BAMN則效果不佳。未來則需要進一步研究來證明這些TuJ1(+)神經元成為成熟神經元的能力及其是否具有電生理活性。
Background: Human neurons are abundant in the brain, however they are difficult to be accessed. Recently, several reports indicate that human neuronal cells can be obtained from the differentiation of induced pluripotent stem cells (iPSCs) or trans-differentiation of induced neurons (iNs) from dermal fibroblasts. The sources of iPSCs or iNs have been demonstrated to be skin fibroblasts, blood cells, hepatocytes or urine cells.
Aims: In this study, we aimed to compare the effects of different neurogenic reprogramming factors, such as SH2B1 (S)、miRNA124 (I)、BRN2 (B)、MYT1L (M)、NeuroD1 (N)、ASCL1 (A) and ATOH1 (AT) on neuronal differentiation efficiency and production of reactive oxygen species in human neural stem/progenitor cells (NSPCs). Furthermore, we aimed to select a useful combination from these neurogenic factors to non-invasively reprogram human urine cells into neuron-like cells.
Results and Conclusion: We found that these neurogenic genes could exhibit differential effects on promoting neural differentiation. Notably, NeuroD1 can generate the highest quantity of TuJ1(+) neurons and lowest quantity of GFAP(+) astrocyte. Furthermore, we found that S-IBM combination can reprogram human fibroblasts into TuJ1 and MAP2(+) neuron-like cells, however, only IBMN and BAMN was demonstrated to directly reprogram the urine cells to TuJ1(+) cells with neuronal morphology, but not S-IBM. Future studies on long-term maturation and the electrophysiological activity of urine-derived TuJ1(+) cells are required to further demonstrate their potential of neuronal maturation and electrophysiological activity.
目錄 1

英文摘要 2

中文摘要 3

縮寫表 4

緒論 5
一、神經系統 5

二、神經系統疾病 5
2.1中樞神經系統疾病 6
2.1.1 神經退化 7
2.1.2 外力撞擊造成 8
2.2周邊神經系統疾病 9
2.2.1運動神經元疾病 9

三、誘導多功能型幹細胞介紹 10
3.1 iPSCs應用於分化神經細胞 11
3.2 從神經疾病患者的纖維母細胞誘導成iPSCs 12
3.2.1利用iPSCs了解帕金森氏症之致病機制 12

四、人類纖維母細胞直接再程式化成為神經細胞 13
4.1 不同的基因產生誘導性神經元 14
4.2 提高形成功能性神經元效率及神經複雜度 14
4.3 p53及hTERT的阻斷提升誘導神經效率 14
4.4 使用尿液細胞直接再程式化為功能性神經元 15

五、實驗材料與方法 16
5.1 細胞株來源 16
5.1.1 化學藥劑 16
5.1.2 螢光染劑 17
5.1.3 抗體 17
5.2 人體試驗委員會(IRB) 18
5.3 尿源細胞收集與培養 18
5.4 慢病毒製作 19
5.5 尿液細胞再程式化為神經細胞 19
5.6 神經幹/前趨細胞(Neural Stem/Progenitor Cells, NSPCs)培養 20
5.7 神經細胞特性基因加速NSPCs分化 20
5.8 免疫螢光染色 21
5.9 流式細胞儀 21

六、實驗結果
6.1 使用神經特性基因加速神經幹/前趨細胞之分化 22
6.2 神經特性基因加速神經幹/前趨細胞測細胞活性氧化物質的含量 22
6.3 尿液細胞再程式化為神經細胞 23
6.4 纖維母細胞進行誘導成神經細胞 23

七.討論 26

八、參考文獻 27

九、表與圖 32
表一、各種神經疾病特定病人來源體細胞建立iPSCs 32
表二、從不同細胞類型直接誘導神經細胞 33
表三、用於IHC神經雙色染色的比例 34
圖一、使用免疫螢光染色確認神經幹/前趨細胞(NSPCs)神經蛋白表現 35
圖二、神經特性基因處理神經幹/前趨細胞(NSPCs),使加速分化 36
圖三、神經幹/前趨細胞(NSPCs)分化五天後,表現MAP2蛋白的神經細胞數目統計 37
圖四、神經特性基因對於神經幹/前趨細胞(NSPCs)分化為神經細胞及星狀膠質細胞之效應 38
圖五、神經幹/前趨細胞(NSPCs)分化五天後,表現GFAP蛋白之星狀膠質細胞數目統計 39
圖六、神經特性基因對於神經幹/前趨細胞(NSPCs)分化過程中,活性氧化物(ROS)產生的效應 40
圖七、神經特性基因對於神經幹/前趨細胞(NSPCs)分化過程中,活性氧化物(ROS)產生的效應 41
圖八、神經幹/前體細胞分化過程蛋白及ROS表現整理表 42
圖九、尿液細胞培養所產生的細胞聚落與型態 43
圖十、將尿液細胞直接誘導形成神經細胞 44







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