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研究生:許奇鴻
研究生(外文):Hsu,Chi-Hung
論文名稱:骨髓間質幹細胞分化為多巴胺神經元蛋白質體學研究
論文名稱(外文):Proteomic Study on Differentiation of Bone-Marrow Mesenchymal Stem Cells into Dopaminergic Neurons
指導教授:李文乾
口試委員:陳文逸王勝仕
口試日期:2011-07-03
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:131
中文關鍵詞:間質幹細胞神經元二維膠體電泳西方墨點法多巴胺
外文關鍵詞:MAPK/ERKCREBPKC-εSWH
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間質幹細胞具體外增生以及跨胚層分化的能力,對於組織病變及退化性疾病的修複有極為廣泛的應用,作為組織工程、細胞治療、再生醫學材料,其發展潛力與突破與日俱增。本研究利用神經幹細胞基礎培養基以及音速波狀蛋白、鹼性成纖維細胞生長因子、成纖維細胞生長因子8、B-27等所組成的誘導培養基進行分化實驗,研究利用SWH訊息路徑剔除之間質幹細胞進行神經誘導分化。使用的工具是二維膠體電泳分析的方法搭配質譜儀分析對蛋白質做身份鑑定。
SWH訊息路徑剔除之間質幹細胞在生長速度以及分化上也比正常間質幹細胞來的快,但在分化過程的型態變化上兩者非常相近的。二維膠體電泳實驗結果顯示,SWH訊息路徑剔除之間質幹細誘導前後比較,找到顯著p<0.01變化且差異量兩倍以上的蛋白質約30點。在鑑定出來的蛋白質中PKC-ε (protein kinase C epsilon)誘導後表現量約上升4倍,PKC-ε在分化過程主要參與MAPK/ERK路徑使神經細胞分化後更趨於成熟,在轉錄調控上有著重要的關係,即是促使重要神經細胞轉錄因子CREB磷酸化。二維膠體實驗也發現Nm23表現量下降約4倍,經過西方點墨法證實Nm23誘導初期表現量上升,但在第誘導2天後開始大量下降,這與我們的細胞型態在第2天時轉變為類似神經細胞型態有關,Nm23在神經分化過程裡在初期主要與神經突觸延伸有關,另外有文獻指出Nm23會抑制MAPK路徑,其與神經分化的過程則尚需進一步釐清。另外動力蛋白Kinesin-1 heavy chain存在於神經與神經膠細胞,其功能可以維持突觸的功能和調節神經可塑性在成熟神經元裡突觸前神經的功能伴演著重要的角色,動力蛋白kinesin-1 heavy chain在誘導5天後表現量約上升7倍。
本研究利用二維膠體電泳發現神經分化前後有差異的蛋白質,在間質幹細胞的分化可能扮演重要的角色。另外在MAPK/ERK訊息傳導路徑的研究,發現ERK抑制劑的存在會使CREB的活化量及BDNF的分泌量減少。ERK的持續活化是神經分化的必須,而且BDNF的分泌量與其接受器TrkB表現量隨分化時間增長而增加。

Mesenchymal stem cells with abilities to proliferate in vitro and differentiate into cells of cross germ layers, accompanying a wide-range of applications in repairing tissue and curing degenerative diseases. The developing potential and breakthrough in tissue engineering, cell therapy, and regenerative medical materials are rising day by day. In this research, we applied tools of two-dimensional gel electrophoresis (2-DE) for protein separation and mass spectrometry analysis for identifying separated proteins in the study of SWH-knockout human mesenchymal stem cells (hMSCs). The SWH-knockout hMSCs were induced to differentiate to neuron-like cells by suing a neural induction medium (NIM) containing SHH, bFGF, FGF8, and B-27.
SWH-knockout hMSCs had much higher growth and differential rates than the normal hMSC. After induction by NIM, both hMSC and SWH-knockout hMSCs have acquired a morphology similar to neural cells. Ten and twenty protein spots were respectively determined to be up- and down-regulated with a significant level of p < 0.01 when the expression of corresponding spots before and after differentiation showed an increase (2-fold) or decrease (0.5-fold). Among the differentially expressed protein spots, three proteins that could be closely related to neurogenesis have been identified by mass spectrometry: Nm23/Nucleoside diphosphate kinase A protein, kinesin-1 heavy chain (UKHC), and protein kinase C epsilon type (PKC-ε). Nm23 was significantly down-regulated, while UKHC and PKC-ε were significantly up-regulated in SWH-knockout hMSCs by the neuronal differentiation. Western blot results indicate that after the induction the Nm23 expression increased initially but decreased gradually at the second day when the cells began to take on a neuron-like morphology. After induction for 5 days, 7-fold increase in UKHC expression and 4-fold increase in PKC-ε expression were observed. UKHC, which is found in nerve and glial cells, functions to maintain synaptic function and regulation of neural plasticity in mature neurons and plays an important role in the presynaptic nerve function. In the differentiation process, PKC-ε could be involved in the MAPK/ERK pathway in the maturation of neuronal cells from differentiating hMSCs.
In summary, these identified proteins that differentially expressed in the 2-DE gels may play important roles in the neural differentiation of hMSCs. In addition, in the study of MAPK/ERK signaling pathway, we found that both the activation of CREB and secretion of BDNF were down-regulated in the presence of ERK inhibitor. A constant level of ERK phosphorylation was required for neural differentiation. Also, the levels of BDNF secretion and TrkB expression increased with the induction time during the differentiating process.

中文摘要 I
Abstract III
目錄 VI
第一章 緒論 1
1.1 前言 1
1.2 間質幹細胞 4
1.3 蛋白質體分析技術 6
1.4 神經幹細胞與神經誘導方式 10
1.4.1 神經細胞分化因子 12
1.4.2 利用化學物質誘導 14
1.5 間質幹細胞誘導成多巴胺神經元 16
第二章 實驗藥品 20
2.1 細胞培養 20
2.2 神經誘導培養基 21
2.3 二維膠體電泳(2-DE) 21
2.4 ELISA 22
2.5 西方墨點法(western blot) 22
2.6 蛋白質定量 24
2.7 SDS-PAGE 24
第三章 儀器設備 25
3.1 二維膠體電泳設備 25
3.2 西方墨點法設備 25
3.3 細胞培養相關設備 25
第四章 實驗步驟與分析方法 27
4.1 實驗流程 27
4.2 間質幹細胞培養與繼代 28
4.2.1 間質幹細胞培養 28
4.2.2 間質幹細胞液態氮筒保存 28
4.2.3 間質幹細胞神經誘導培養基 29
4.3 二維膠體電泳(2-DE) 29
4.3.1 胞內蛋白質前處理 29
4.3.2 蛋白質定量 30
4.3.3 回溶蛋白質等電點分離(IEF) 31
4.3.4 配置SDS-PAGE(2-DE) 33
4.3.5 SDS-PAGE電泳 34
4.3.6 銀染法(硝酸銀法) 35
4.3.7 蛋白質樣品處理(In-Gel Digestion) 35
4.3.8 蛋白質身份鑑定 37
4.3.9 ImageMaster軟體使用 37
4.4 西方墨點法(western blot) 38
4.5 Enzyme-Linked Immunosorbent Assay (BDNF kit) 42
4.6 Enzyme-Linked Immunosorbent Assay 43
(Dopamine kit) 43
第五章 實驗結果與討論 45
5.1 SWH路徑剔除之間質幹細胞 45
5.2 SWH路徑剔除之間質幹細胞型態變化 47
5.3 二維膠體電泳定量與分析 49
5.4 SWH路徑剔除間質幹細胞比對 50
5.5 SWH路徑剔除間質幹細胞神經分化差異表現之蛋白質 57
5.6 西方墨點法驗證已身份鑑定之蛋白質 63
5.7 神經分化細胞的Dopamine分泌量 67
5.8 神經分化過程訊息傳導蛋白質的變化 68
6.1結論 76
6.2 建議 78
參考文獻 79
附錄A 二維膠體電泳重覆實驗結果 93
附錄B 蛋白質身份鑑定結果 103
附錄C 二維膠體電泳膠片分析結果差異表現之蛋白質點 108
附錄D 西方墨點法重覆實驗 112
附錄E 西方墨點法驗證已身份鑑定之蛋白 120


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