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研究生:蘇育世
研究生(外文):Yu-Shih Su
論文名稱:硬膜外移植誘導性萬能幹細胞分化之神經先驅細胞促進中風成鼠的功能恢復
論文名稱(外文):Functional improvement following epidural transplantation of iPSC-derived neural progenitors in adult murine stroke
指導教授:李怡慧李怡慧引用關係
指導教授(外文):I-Hui Lee
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:腦科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:73
中文關鍵詞:誘導性萬能幹細胞神經幹細胞中風
外文關鍵詞:induced pluripotent stem cells (iPSCs)neural stem cells (NSCs)stroke
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由纖維母細胞來的誘導性萬能幹細胞可提供客製化的細胞治療來源,但腫瘤風險及異常免疫反應是轉譯應用的最大阻礙。本論文包含兩項研究主題:(1) 探討成年中風大鼠以硬膜外移植誘導性萬能幹細胞分化之神經前驅細胞的療效及可能的治療機制;(2)了解中風後內生性的皮質神經生長機制。大鼠於中大腦動脈阻塞術後7天,透過局部硬膜外纖維凝膠移植人類誘導性萬能幹細胞分化之神經先驅細胞,並比較移植組及對照組行為和組織學上的差異。另藉由體外和缺氧缺糖的皮質神經細胞隔膜共同培養的模型,比較誘導性萬能幹細胞分化之神經先驅細胞和其他人類幹細胞,對皮質神經生長的影響,並以系統生物學的方式分析其富含的分泌激素和可能的分子機制。此外,利用皮質第五層錐狀神經元帶有黃色螢光的轉殖基因小鼠作中大腦動脈阻塞實驗,分析其內生性的皮質神經再生與功能恢復。經過體外的神經分化後,大於80%的誘導性萬能幹細胞表達PAX6+/SOX1+等代表神經先驅細胞的蛋白。移植此神經先驅細胞到中風大鼠約10天後,發現癱瘓前肢的使用比例及握力開始較對照組有顯著且持續的進步;於移植後21天的組織學分析顯示,移植組的腦梗塞及萎縮體積降低、發炎反應和細胞凋亡減少,血管新生增加、寡樹突前驅細胞及白質徑路完整性增加。硬膜外移植的神經先驅細胞移行至梗塞皮質周圍,僅剩少量的存活且無腫瘤形成。在隔膜共同培養的實驗中顯示,誘導性萬能幹細胞分化之神經先驅細胞與來自骨髓或臍帶瓦頓氏凝膠的間葉幹細胞相比,其分泌的特殊激素可促進神經生長及抑制星狀膠細胞增生。在Thy1-YFP轉殖基因小鼠的中風實驗,隨著癱瘓肢的功能恢復,梗塞皮質周邊的錐狀神經元的軸/樹突、胼胝體的神經徑路、及皮質紅核徑路,皆於中風後逐漸增生。硬膜外移植誘導性萬能幹細胞分化之神經先驅細胞可減少腫瘤風險及異常免疫反應,並透過分泌效果促進內生性的組織修復,進而促進中風後功能的恢復。進一步釐清其治療的分子機轉,將有助於發展中風後藥物及細胞治療。

Induced pluripotent stem cells (iPSCs) derived from fibroblasts are promising for tailor-made cell therapy; however, tumorigenesis and immunogenicity hamper their translational implantation. We have two aims in this thesis: (1) to investigate the therapeutic effects and molecular mechanism following epidural transplantation of iPSCs-derived neural progenitor cells (iPSC-NPCs) in adult stroke rats; (2) to study endogenous cortical neurite outgrowth after stroke. Human iPSC-NPCs or vehicle were delivered via epidural fibrin glue following permanent middle cerebral artery occlusion (MCAO) to compare behavioral and histological changes in rats. A transmembranous co-culture system with rat cortical cells subjected to oxygen-glucose deprivation (OGD) was used to characterize the iPSC-NPC secretome compared with different human stem cell types using a systems biology approach including cytokine arrays and whole-genome microarrays. Besides, Thy1-YFP mice with fluorescent cortical layer V pyramidal neurons were utilized to characterize the post-stroke endogenous cortical neurite outgrowth in relation to functional recovery.Over 80% Pax6+/Sox1+ iPSC-NPCs were obtained after differentiation and mechanical enrichment. The iPSC-NPC-transplanted rats showed significant improvement in their paretic forelimb usage and grip strength from 10 days post-transplantation (dpt) onwards compared with the vehicle-transplanted rats. At 21 dpt, the cell-treated rats had significantly reduced infarct/atrophy volumes, ameliorated microglial infiltration, astrogliosis, and apoptosis, as well as augmented angiogenesis, oligodendrocyte progenitors, and white matter tract integrity. Grafted iPSC-NPCs migrated into peri-infarct coetex but poorly survived with no observed tumors. In the transmembranous co-cultures, the iPSC-NPCs, in contrast to mesenchymal stem cells from bone marrow and umbilical cord Wharton’s jelly, enhanced neuronal growth and mitigated astrogliosis via a unique secretome. On the other hand, Thy1-YFP stroke mice had temporally functional improvements in association with increased neurite density in the peri-infarct cortex and the corticorubral tract after stroke. These results suggest that the epidural delivery of iPSC-NPCs may not only decrease tumorigenic and immunogenic risks but also enhance functional improvement after stroke through bystander stimulation of endogenous regenerative response. Further mechanistic studies can shed light on pharmacological or cell therapy after strok.

目錄
論文審定同意書 II
論文電子檔著作權授權書 III
致謝 IV
中文摘要 VI
英文摘要 VIII
縮寫對照 X
目錄 XII
表目錄 XV
圖目錄 XVI
第一章 研究背景 1
第一節 中風後神經再生與可塑性 1
一、內生性神經幹細胞新生 1
二、皮質神經軸突新生 1
第二節 幹細胞移植療法在中風的應用 2
一、幹細胞的種類 2
二、臨床的幹細胞治療試驗 4
第三節 誘導性萬能幹細胞及衍生的神經幹/先驅細胞 5
第四節 神經幹/先驅細胞移植對中風的治療 7
一、幹細胞的移植時機 7
二、幹細胞的移植途徑 8
三、幹細胞的治療機制 8
第二章 研究假設與目的 10
第三章 研究方法 11
第一節 實驗設計 11
第二節 人類誘導性萬能幹細胞的培養與神經分化 12
第三節 人類骨髓和臍帶的間葉幹細胞培養 13
第四節 中大腦動脈阻塞手術和中風後細胞移植 13
第五節 行為分析 14
一、握力測試 14
二、圓筒內前肢使用率測試 15
三、滾筒跑步機 15
四、通心粉矩陣抓取測試 15
第六節 組織學分析 16
第七節 皮質神經細胞的缺氧缺糖模式與幹細胞隔膜共同培養 17
第八節 即時定量聚合酶連鎖反應分析 18
第九節 基因微陣列表達分析 19
第十節 細胞素陣列表達分析和西方墨點法 20
第十一節 Thy1-YFP轉殖基因鼠在中風後的神經生長分析 21
第十二節 統計分析 21
第四章 研究結果 23
第一節 人類誘導性萬能幹細胞分化成神經先驅細胞 23
第二節 神經先驅細胞移植促進中風後功能恢復並減少腦梗塞及萎縮 24
第三節 神經先驅細胞移植增加內生性的修復 24
第四節 神經先驅細胞在隔膜共同培養模型中分泌的特殊激素 25
第五節 神經先驅細胞改變缺氧缺糖皮質細胞的基因和蛋白質表達 27
第六節 中風後皮質錐狀神經元和內生性神經幹細胞的生長與行為恢復 28
第五章 討論 30
第一節 本研究的主要發現與意義 30
一、硬膜外移植為有效的移植途徑 30
二、移植神經先驅細胞增加梗塞周邊的內生性寡樹突先驅細胞 31
三、神經先驅細胞的分泌激素與影響 32
第二節 本研究的限制與未來研究方向 34
一、神經先驅細胞治療機制的驗證 34
二、移植細胞的存活與分化 34
三、中風後皮質錐狀神經元的純化及轉錄體分析 35
第六章 結論 36
參考文獻 37
附表 48
附圖 58
附錄-投稿著作 74

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