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系統識別號 U0026-2606201714023200
論文名稱(中文) 纖維母細胞生長因子九於脂肪幹細胞進行神經球生成過程中所扮演的角色
論文名稱(英文) Role of Fibroblast Growth Factor 9 in Neurosphere Formation of Adipose-derived Stem Cells
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 105
學期 1
出版年 106
研究生(中文) 陸詩羽
研究生(英文) Shih-Yu Lu
學號 T96034036
學位類別 碩士
語文別 中文
論文頁數 75頁
口試委員 指導教授-吳佳慶
口試委員-黃步敏
口試委員-孫孝芳
口試委員-莊季英
口試委員-楊尚訓
中文關鍵字 脂肪幹細胞  纖維母細胞生長因子九  纖維母細胞生長因子受器  神經細胞球  許旺細胞 
英文關鍵字 Adipose-derived stem cell  fibroblast growth factor 9  FGF receptors  neurosphere  Schwann cell 
學科別分類
中文摘要 脂肪幹細胞 (adipose derived stem cell, ASC) 可從脂肪組織中取得並為細胞療法中重要的幹細胞來源,可分化成神經導向細胞 (neural lineage cell, NLC) 促進損傷後神經再生。纖維母細胞生長因子九 (fibroblast growth factor 9, FGF9) 可以改變幹細胞的增殖及細胞發展。在本論文中,目的是探討神經球形成過程中FGF9的作用,在有無FGF9刺激下以甲殼素 (chitosan) 塗佈之培養皿培養ASC誘導分化成NLC,其中的基因表現以聚合酶連鎖反應 (polymerase chain reaction, PCR) 檢測,蛋白質表現則以西方墨點法和螢光染色觀察,並以訊息傳遞路徑抑製劑或shRNA探究Akt訊息傳遞路徑在神經細胞球形成期間的影響。結果表示,ASC形成的神經細胞球含有NLC和神經膠質細胞,FGF9可誘導神經細胞球往神經膠質細胞分化。Akt、細胞外調節蛋白 (extracellular regulated protein, ERK) 訊息傳遞路徑活化在形成神經細胞球後下降,對於神經細胞球生成而言,訊息傳遞路徑失活化為必備因素。當神經細胞球形成期間加入FGF9會短暫增加Akt訊息傳遞路徑的活化,並且透過Akt抑制劑阻斷Akt訊息傳遞路徑證實Akt訊息傳遞參與在細胞球分化過程中。另外以纖維母細胞生長因子受器二與三 (fibroblast growth factor receptor 2 and 3, FGFR2 and 3) 阻礙抗體與FGFRs之小髮夾核醣核酸 (short hairpin RNA of FGFRs, shFGFRs) 阻礙FGFRs與FGF9作用,觀察神經細胞球形成期間FGF9的主要的受器。當阻礙FGFR2或FGFR2生成阻斷,原由FGF9導致表現提高的NLC標的基因,表現會因此下降。以上結果表示,在神經細胞球生成期間FGF9藉由與FGFR2作用並通過Akt訊息傳遞路徑影響NLC分化。當前研究的發現可能在神經細胞球形成期間可通過調節FGF9含量控制ASC分化成所需的神經譜系細胞。
英文摘要 Adipose-derived stem cells (ASCs) were an ideal cell source for stem cell therapy can promote nerve regeneration after injury. The fibroblast growth factor 9 (FGF9) can alter the proliferation and cell-fate decision in stem cells. In my project, we aim to investigate the role of FGF9 during neurosphere formation of ASCs. We used chitosan- coated dish to culture ASCs to induce sphere formation with or without addition of FGF9. The expression of gene was detected by PCR and the protein expression level was observed by Western blot and IF staining. Besides, we investigated the involvement of potential signaling pathway of FGF9 signaling during the sphere formation using specific inhibitors or shRNA. The results showed the spheres formed by ASCs containing both neuronal and glial lineage cells. Addition of FGF9 induced spheres differentiation into Schwann lineage cells. Then, the activation of Akt, ERK signal pathway was decreased after forming the sphere and the inactivation of signal pathway was necessary for sphere formation. However, the phosphorylation of Akt pathway was transiently preserved by FGF9. Furthermore, the involvement of Akt signaling was confirmed by blockage of Akt signaling. The FGFR2 and 3 blocking antibody and shFGFRs was used to block FGFRs to investigate the major FGFR of FGF9 induced Schwann cell differentiation. The expression of Schwann cell markers induced by FGF9 was decreased when knockdown FGFR2. The result suggested that FGF9 could modulate NLC differentiation by activating Akt signaling via binding with FGFR2 during sphere formation. The finding of current study may show the possibility of differentiating ASCs into desired neural lineage by modulating the level of FGF9 during sphere formation.
論文目次 中文摘要 I
英文摘要 III
目錄 XIII
表目錄 XVI
圖目錄 XVII
第一章 前言 1
1.1 神經受損與再生 1
神經受損 1
瓦勒氏退化 1
軸突再生 2
神經再支配 3
神經損傷治療 5
斷端縫合與神經束縫合 6
神經移植 6
神經導管接合 6
細胞治療 8
1.2 脂肪幹細胞 10
1.3 纖維母細胞生長因子九 11
研究目的 12
具體目標 12
第二章 材料與方法 13
2.1 脂肪幹細胞培養 13
2.2 培養表面之甲殼素塗層處理 14
2.3 細胞球生成 16
2.4 RNA萃取與逆轉錄聚合酶鏈式反應 17
核醣核酸萃取 17
逆轉錄反應 17
聚合酶鏈式反應 18
2.5 西方墨點分析法 22
蛋白質養本收取 22
凝膠電泳 22
轉漬 22
阻攔作用 23
2.6 免疫螢光染色 28
貼附細胞 28
細胞球 28
2.7 DNA質體純化 31
菌株挑選與菌液培養 31
小量製備 31
中量製備 31
2.8 電穿孔 35
2.9 BrdU細胞增殖分析 36
2.10酶聯免疫吸附測定 38
測定盤製備 38
酶聯免疫吸附測定 38
第三章 實驗結果 40
3.1觀察脂肪幹細胞形成神經球過程中神經導向基因與蛋白質的表現 40
3.2 觀察纖維母細胞生長因子九與纖維母細胞生長因子受器於脂肪幹細胞及神經導向細胞中基因與蛋白質表現 42
3.3 纖維母細胞生長因子九對神經球大小之影響 44
3.4 纖維母細胞生長因子九對神經導向細胞分化的影響 46
3.5探討纖維母細胞生長因子九在細胞球生成過中所參與的訊息路徑 49
3.6 訊號路徑磷酸化對細胞球生成的重要性 51
3.7 FGFR2為FGF9主要受器以活化下游訊息途徑與影響NLC分化 56
第四章 討論 62
第五章 結論 66
第六章 參考文獻 68

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