幹細胞具有長期自我增生與分化成特定型態與功能的細胞，因此幹細胞在發育生物學、基因體學、基因轉殖方法、臨床應用潛能、基因治療與組織工程上均是非常重要的研究工具，此具有分化多能性的幹細胞是功能性退化疾病的重要細胞來源。因此，本研究之目的係以外源性綠色螢光基因（green fluorescent protein，GFP）對豬胚幹細胞進行基因轉殖，建立可供移植後追蹤之豬胚幹細胞株，並建立豬胚幹細胞之神經分化定向誘導技術，應用於大鼠帕金森氏症疾病模式之治療應用。雖然從畜產動物分離具有分化多能性的胚幹細胞株較小鼠困難，然本研究已順利自著床前的囊胚成功建立豬胚幹細胞株，並且利用電穿孔法（electroporation）進行豬胚幹細胞之基因轉殖，將外源性GFP成功轉殖入豬胚幹細胞，轉殖後之豬胚幹細胞表現胚幹細胞分化多能性專一細胞標記如Oct-4、AP、SSEA-4、TRA-1-60與TRA-1-81等，顯示經轉染後之豬胚幹細胞株仍保有分化多能性。利用促進神經分化之因子，如維生素A酸（retinoic acid，RA）、音速小子（sonic hedgehog，SHH）與纖維母細胞生長因子（fibroblast growth factor，FGF）進行神經定向誘導分化處理後之衍生細胞，可表現神經專一性抗體如nestin、NFL、MAP2、GFAP、A2B5、TH、ChAT與GABA等，顯示豬胚幹細胞具分化成為神經細胞之潛能。而將此株攜有GFP豬胚幹細胞移植於大鼠腦部，以非侵入式活體影像分析系統（IVIS 50）與侵入式活體影像分析系統（Cellvizio®），追蹤移植後之豬胚幹細胞在大鼠腦部存活與生長情形。在持續3個月的追蹤期間，均可於大鼠腦部測得螢光反應，且處理組大鼠腦組織的螢光表現量比對照組高出2倍以上，顯示攜有GFP豬胚幹細胞確實可做為移植後之追蹤標的細胞。而將豬胚幹細胞及其神經定向誘導分化後之衍生細胞移植於大鼠帕金森氏症疾病模式，移植豬胚幹細胞者於1個月時可見到功能性行為的回復，然第2與3個月時則功能性行為回復較不明顯；而移植誘導化後之成熟神經元細胞者於第3個月時，帕金森氏症大鼠之旋轉行為則有明顯降低，顯示具有治療帕金森氏症之效果，移植部位未有腫瘤之生成，且經免疫染色後發現呈現TH與DA陽性反應，顯示具有多巴胺生成性神經元（dopaminergic neuron）。此等研究結果顯示，所建立之攜有外源性GFP之豬胚幹細胞株，不僅可供胚幹細胞與其衍生細胞應用於活體移植試驗之後續追蹤與研究，並可進一步藉由運用豬胚幹細胞於神經損傷與退化之研究，開發新療法與治療模式，進而得到人類胚幹細胞研究上所不易取的重要資訊，極具應用價值。

Stem cells have the ability to reproduce themselves for a long period and differentiate into specific morphological and functional cells. The stem cells are an important material in the developmental biology, genomics, and transgenic methods, as well as in potential clinical applications, gene therapy and tissue engineering. The pluripotent stem cells will be a valuable source in numerous functional degenerated pathologies. Therefore, the objective of this research program was to establish transgenic porcine embryonic stem (pES) cell lines which can express green fluorescent protein (GFP) report gene stably for tracking after transplantation. We also developed a directed differentiation of pES into neural lineages and applied in rat Parkinson’s disease model. Although the establishment of pluripotent ES cell lines from domestic species is much more difficult than that in murine species, our results had successfully isolated and established pES cell lines from pre-implantation blastocysts. Furthermore, we established the novel GFP-expressing pES cell lines (pES/GFP+), which were obtained by electroporation- mediated transfection with exogenous GFP gene. These pES/GFP+ cells exhibited pluripotent markers including Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81 as that of human ES cells. The strategy of directed neural differentiation was to culture pES with neurogenic stimulators such as retinoic acid (RA), sonic hedgehog (SHH), and fibroblast growth factor (FGF). Upon directed differentiation toward neural differentiation, these pES-derived cells exhibited typical neuronal morphology and expressed neural lineage-specific markers such as nestin, NFL, MAP2, GFAP, A2B5, TH, ChAT, and GABA. These results showed the pES cells had the potential to differentiate into neural lineages. When pES/GFP+ cells were transplanted into the SD rat’s brain, and their survival and development was determined by the non-invasive In Vivo Imaging System (IVIS 50), and the invasive fibered confocal Cellvizio® Imaging System (Cellvizio®). The results showed that fluorescent signals from pES/GFP+ cells on the injection site of SD rats’ brain could be detected through the experimental period of 3 months. The level of fluorescent signals detected in treatment groups was two folds above that of the control group. Besides, the functional behavior recovery analysis by amphetamine-induced rotation test indicated the PD rat grafted with pES/GFP+ cells and their derived neural progenitors showed no significant recovery of rotation rate in these two treatments because a progressively increased relative rotation through 3 months duration. However, the relative rotation of PD rats grafted with the pES/GFP+-derived mature neurons, showed a stably decrease relative rotation and resulted in a functional recovery from Parkinsonian behavioral defects. Following 3 months completion of behavioral analyses, PD rats were sacrificed for immunohistochemical analysis. In the section of injected site without tumorgenesis and showed the survival and dopaminergic differentiation of grafted pES/GFP+ derived cells when stained with anti-TH and anti-DA. To our knowledge, there have been no reports of establishing GFP-expressing pES cell lines. These novel pES/GFP+ cell lines established in this study might serve as a non-rodent model and could benefit to the studies involving ES cell transplantation, cell replacement therapy, tissue regeneration, and actual approach for pre-clinical research due to their traceable capacity.

Abstract
Chinese ---------------------------------------------------------1
English -----------------------------------------------------------3

Chapter 1: Introduction
Introduction ------------------------------------------------------6
References ----------------------------------------------------12

Chapter 2 : Establishment and Characterization of Novel Porcine Embryonic Stem Cell Lines Expressing hrGFP
Abstract -----------------------------------------------------------22
Introduction ------------------------------------------------------23
Materials and Methods ---------------------------------------25
Results -----------------------------------------------------------33
Discussion ------------------------------------------------------35
References ------------------------------------------------------39
Figures and Tables -------------------------------------------44

Chapter 3: Directed Differentiation of Porcine Embryonic Stem Cells into Neural Lineages
Abstract -----------------------------------------------------------52
Introduction ------------------------------------------------------54
Materials and Methods ---------------------------------------56
Results -----------------------------------------------------------60
Discussion ------------------------------------------------------63
References ------------------------------------------------------67
Figures and Tables -------------------------------------------75

Chapter 4: Transplanted Performance and Therapeutic Potential of Porcine Embryonic Stem Cells in Parkinson’s Disease Model
Abstract -----------------------------------------------------------82
Introduction ------------------------------------------------------83
Materials and Methods ---------------------------------------86
Results -----------------------------------------------------------91
Discussion ------------------------------------------------------93
References ------------------------------------------------------96
Figures and Tables -----------------------------------------104

Appendix
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