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。

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.

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