胎盤源多功能細胞分化成類神經細胞之訊息傳導途徑研究

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姓名

吳家安(Jia-an Wu) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

胎盤源多功能細胞分化成類神經細胞之訊息傳導途徑研究
(Elucidate the signaling pathway of PDMCs (Placenta-Derived Multipotent Cells) differentiated into Neuron-like cells.)

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摘要(中)

近年來幹細胞的研究於再生醫學上掀起一股熱潮，而常見用於治療的骨髓幹細胞及胚胎幹細胞卻存在著具侵入性取得及道德上的爭議，突顯出開發替代的治療細胞極為重要。而胎盤源多功能細胞(Placenta-Derived Multipotent Cells, PDMCs)具有分化成脂肪細胞、成骨細胞、肝細胞及神經細胞等優越分化能力，且取得來源不具道德的問題，是較佳的成體間葉幹細胞來源。
由於蛋白質磷酸化迅速及可逆的調控特性，能夠即時地反應細胞受到刺激時所產生的訊息傳遞情形。在先前研究指出，胎盤源多功能細胞受3-異丁基-1-甲基黄嘌呤(IBMX)刺激後可分化成神經細胞，因此本研究嘗試利用磷酸化蛋白質體學技術，研究當PDMC細胞受到IBMX刺激時，細胞內所誘發的蛋白質磷酸化變異及其中訊息傳遞路徑的調控，並探討可能的生理意義。首先利用immobilized metal afﬁnity chromatography (IMAC)純化細胞中的磷酸化蛋白，再經由二維膠體電泳分析，並將這些受藥物刺激後磷酸化的蛋白質進行質譜儀進行鑑定再輔以文獻分析，推測出可能的訊息傳遞網絡。再透過西方墨點法的驗證，顯示IBMX的刺激確實會透過磷酸化調控的方式，以PKC/ERK/c-Jun的路徑進行調控，可能是藉由此訊息傳導路徑促進胎盤源多功能細胞分化成神經細胞，而其他如細胞形態改變及細胞骨架的重組可能也有其他的訊息傳導路徑參與。未來可望針對這些結果做更進一步的研究以找出IBMX刺激後所制動的訊息傳導路徑及其啟動之基因，並以這些資訊來幫助胎盤幹細胞專一性的分化成特定的神經細胞並應用於臨床治療。

摘要(英)

In recent years, the research of regenerative medicine focus on the studying and utilizing stem cells. Current sources of stem cells include embryonic stem cells (ESCs) and adult stem cells (ASCs). However, there are numerous ethical concerns exist in ESCs and as to ASCs, which usually obtained from the bone marrow through an invasive procedure. The placenta-derived multipotent cells (PDMCs) are an ethically uncontroversial source, and the cells exhibit the ability to differentiate into many cellular types, including osteoblasts, adipocytes, hepatocytes, and neurons. Therefore, PDMCs could be the considerable alternative resources of mesenchymal stem cell.
It has been found that under 3-isobutyl-1-methylxanthine (IBMX) inductions, the PDMCs were capable of differentiating into neural-like cells; however, the signal transduction scenario and molecular mechanism behind the phenomenon are still unknown, therefore, we attempts to study this process by phosphoproteomic approaches. After phosphoprotein enrichment with IMAC from IBMX induced PDMCs, two dimensional gel electrophoresis following LC/MS-MS identification were enrolled and the phosphorylated proteins specific induced by IBMX were identified and analyzed by the signal transduction database tool.
After Western blot validation and literature searches, we found that IBMX would induce phosphorylation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun and their sequential phosphorylation might involve in the signal transduction in the neural cell differentiation. Besides, cell morphology changes would also be induced through dephosphorylation of other proteins such as Heat Shock Protein 27 (HSP27). However, the phosphorylation regulations and their physiological roles need to be further validated. The information provide by this thesis may help us decipher the mechanism of neuron differentiation and may contribute to the neural degenerative disease treatment in the future.

關鍵字(中)

★ 胎盤源多功能細胞
★ 訊息傳導
★ 神經誘導分化

關鍵字(英)

★ PDMC
★ neural differentiation
★ signal transduction

論文目次

摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 ix
表目錄 ix
第一章緒論 1
第二章文獻回顧 2
2.1 幹細胞 2
2.1.1 幹細胞研究背景 2
2.1.2 何謂幹細胞 3
2.1.3 幹細胞的分類 4
2.1.4 幹細胞的來源 5
2.1.5胎盤源多功能細胞(placenta-derived multipotent cells,PDMCs) 7
2.1.6 胎盤源多功能細胞的神經誘導分化 8
2.2 細胞訊息傳遞 9
2.2.1 細胞訊息傳遞系統的基本特徵 10
2.2.2 神經幹細胞分化成神經寡突細胞之訊息傳導路徑 11
2.3 磷酸化蛋白質體學 14
2.3.1 蛋白質體學 (Proteomics) 14
2.3.2 磷酸化蛋白質介紹 16
2.3.3 磷酸化分子純化 19
2.3.3.1 免疫沉降法 20
2.3.3.2 親和純化法 21
2.3.3.3化學修飾法 24
2.3.4 質譜分析 24
2.4 研究目的 25
第三章材料與方法 26
3.1 實驗設備 26
3.2 實驗藥品 27
3.3 實驗方法 29
3.3.1 細胞培養技術 29
3.3.2 免疫組織化學染色(Immunohistochemistry,IHC) 31
3.3.2 蛋白質定量分析 32
3.3.3 磷酸化蛋白純化 33
3.3.4 二維膠體電泳法 (two-dimensional electrophoresis; 2-DE) 34
3.3.5 蛋白質膠內酵素水解(In-gel digestion) 39
3.3.6 蛋白質溶液內酵素水解(In-solution digestion) 40
3.3.7 蛋白質身分鑑定 40
3.3.8 西方墨點分析法（Western Blotting Analysis） 41
第四章結果與討論 45
4.1 細胞型態的觀察及免疫組織化學染色(IHC) 45
4.2 磷酸化蛋白的純化 46
4.3 二維膠體電泳分析 48
4.3.1 純化後的磷酸化蛋白於二維膠體電泳分佈 48
4.3.2 不同誘導時間經IMAC純化磷酸化蛋白後的二維電泳膠圖 49
4.3.3 二維膠體電泳結果分析 53
4.4 西方墨電法驗證 56
4.5 添加PKC及ERK的抑制劑測試結果 60
4.6 推測可能的訊息傳導途徑: 66
4.6.1 PKC/ERK/c-Jun 66
4.6.2 HSP27/cytoskeleton protein 68
第五章結論與建議 70
參考文獻 72
附錄液相層析串聯式質譜(LC-MS/MS)與SwissProt資料庫比對結果 80
附錄溶液內酵素水解結果比對KEGG MAPK pathway圖 88
附錄與ERK1與ERK2具有交互作用之基因差異 89

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指導教授

陳文逸(Wen-yih Chen)

審核日期

2012-1-18

推文