臺灣博碩士論文加值系統

帕金森氏症是一種神經退化性疾病，主要是由於患者腦中黑質紋狀體的多巴胺神經元不正常的死亡，而造成腦中神經傳導物質多巴胺的分泌量下降所引起的疾病。隨著幹細胞的研究進展，以細胞治療的方式替補腦中死亡細胞的功能成為可應用的治療策略。
根據文獻指出，人類羊水幹細胞具有分化為多種間質譜系細胞及類神經細胞之能力。此類細胞存有一特殊群落，在神經誘導分化前後，皆可表現神經相關蛋白如NES、TUBB3、NEFH、NEUNA60、GALC和GFAP，被認為具有較佳神經分化潛能，而本研究中命名其為羊水幹細胞-N (AFSC-N)。本研究擬利用六-羥基多巴胺建立的類帕金森氏症大鼠模式探討以AFSC-N進行細胞治療之可行性。首先將AFSC-N移植入類帕金森氏症大鼠右側大腦紋狀體，並在術後，藉誘導行為測試、免疫組織染色觀察大鼠多巴胺神經恢復狀況。本實驗結果得知，類帕金森氏症大鼠在接受腦部細胞移植後，有七成的大鼠在治療期，受阿普嗎啡誘導的向健側旋轉行為逐漸改善，在12週可降低79%，另有將近三成的大鼠有初期行為改善但在12週後呈現惡化的現象，而sham組之向健側旋轉行為則持續惡化，至12週上升約59%。藉由腦切片組織之免疫染色發現，經AFSC-N移植的右側大腦紋狀體不僅可表現人類粒線體特異蛋白，還會表現多巴胺神經相關蛋白如多巴胺運送蛋白與酪胺酸羥化酶。由此可知，AFSC-N不僅可在大鼠腦中存活，並有分化為多巴胺神經細胞的潛能，其移植有利於類帕金森氏症大鼠的行為恢復。由此可預期AFSC-N在帕金森氏症未來的臨床治療上具有相當潛力。

Parkinson’s disease is a neurodegenerative disease characterized by the loss of mesencephalic dopaminergic neuron in the substantia nigra pars compacta and the concomitant reduction of dopamine in the striatum. According to previous study, amniotic fluid-derived stem cells (AFSC) exhibit the capacity to differentiate into multiple mesenchymal lineages and neuron-like cells. Furthermore, one sub-population of AFSC, designated as AFSC-N, expresses some neural markers such as NES, TUBB3, NEFH, NEUNA60, GALC, and GFAP both before and after neural induction. The overall objective of this study is to investigate the feasibility of using these cells for the cell therapy of Parkinson’s disease by a parkinsonian rat model.
The injections of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle of male Sprague-Dawley rats were performed to establish the parkinsonian animal model. After 4 weeks, AFSC-N cells were transplanted in the striatum of the parkinsonian rats. The effect of cell therapy was evaluated by analyzing the recovery in rotation behavior and the expression of the related proteins in the brain tissue by immunohistochemistry and immunofluorescence. The experimental results showed that 72% of the 6-OHDA lesioned rats with transplantation of AFSC-N cells resulted in decreased apomorphine-induced rotations by 52%, 60%, 58% and 79% after the transplantation of 3, 6, 9 and 12 weeks respectively; however the rotation behavior of the other 28% rats with AFSC-N transplantation became worse at the 12 weeks after transplantation. As a control, the apomorphine-induced rotations of the sham group increased 59% after 12 weeks. The immunohistostains on the sectioned brain tissue showed that the transplanted cells which expressed the human specific mitochondrial proteins were found to express the tyrosine hydroxylase and dopamine transporter. The cells could not only survive in the brain of the parkinsonian rat but also differentiate into dopamine neurons. Based on these results, it is suggested that the transplantation of AFSC-N cells was beneficial to the recovery of the experimental parkinsonian rats.

中文摘要……………………………………………………………i
英文摘要…………………………………………………………ii
目錄……………………………………………………………………...iii
圖目錄…………………………………………………………………...vi
表目錄………………………………………………………………….viii
一、研究背景及目的……………………………………………………..1
二、文獻回顧……………………………………………………………..3
2.1、帕金森氏症……………………………………………………….3
2.2、帕金森氏症的病因…………………….…………………………3
2.2.1、遺傳上的缺失造成帕金森氏症……………….…………….3
2.2.2、原發性的帕金森氏症………………………………………..5
2.3、多巴胺…………………………………………………………….5
2.4、治療策略………………………………………………………….6
2.4.1、症狀舒緩策略…………………………..……………………6
2.4.1.1、藥物治療………………………...………………………6
2.4.1.2、手術治療………………………...………………………7
2.4.2、細胞治療策略…………………………..……………………8
2.4.2.1、胚胎腦移植治療…………………...……………………8
2.4.2.2、幹細胞移植治療………………………………………...9
2.4.2.2.1、神經幹細胞…………………………………………9
2.4.2.2.2、胚胎幹細胞…………………..……………………10
2.4.2.2.3、間質幹細胞…………………..……………………11
2.5、人類羊水間質幹細胞……………………...……………………12

三、研究方法……………………………………………………………13
3.1、實驗設計原理…………………………………………………...13
3.1.1、六-羥基多巴胺……………………...………………………13
3.1.2、阿普嗎啡……………………………………………………14
3.1.3、免疫組織化學(螢光)染色………….………………………14
3.2、實驗方法…………………………………...……………………15
3.2.1、誘導藥物配製-多巴胺神經毒素-六-羥基多巴胺…………15
3.2.2、建立類帕金森氏症動物模式………………………………15
3.2.3、動物來源及飼養方式………………………………………17
3.2.4、人類羊水幹細胞-N的培養方式….…..……………………17
3.2.5、細胞繼代方法………………………………………………17
3.2.6、阿普嗎啡誘導大鼠行為測試………………………………18
3.2.7、細胞的移植方法……………………………………………18
3.3、治療結果評估…………………………...……………………20
3.3.1、類帕金森氏症大鼠的行為恢復評估……………………20
3.3.2、統計分析…………………………………………………20
3.3.3、大腦組織切片……………………………………………20
3.3.4、免疫化學染色……………………………………………21
3.3.5、免疫螢光染色……………………………………………22
四、實驗結果……………………………………………………………25
4.1、阿普嗎啡誘導治療後類帕金森氏症大鼠的行為測試………...25
4.2、免疫染色………………………………………………………...31
4.2.1、酪胺酸羥化酶免疫化學染色………………………………31
4.2.2、免疫螢光染色••••••••••••••••••33
五、討論…………………………………………………………………37
六、結論…………………………………………………………………41
七、參考文獻……………………………………………………………43

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