臺灣博碩士論文加值系統

目前已知酸性纖維母細胞成長因子(acidic Fibroblast Growth Factor，aFGF)是一種存在於中樞神經系統之神經滋養分子，在受傷的脊髓組織中可以促進神經細胞的存活與突觸的生長。但是酸性纖維母細胞成長因子(aFGF)是藉由何種機轉來執行神經保護功能，目前尚待研究。依據本實驗室先前研究成果顯示，當注射酸性纖維母細胞成長因子(aFGF)於脊髓損傷大鼠中，可以改善其後肢運動的功能。本研究利用蛋白質體學與生物資訊學的方式，分析鑑定在損傷後第二十八天之正常組(sham group)、脊髓損傷組(SCI group)與加藥組(aFGF treated group)之間的二維蛋白質電泳圖譜，結果發現二十五個蛋白質之表現量有顯著差異。根據生物資訊學方法的分析結果，可以將這二十五個蛋白質分類為五種不同的蛋白質表現型態，且分屬於五種不同的生物功能類別，包含細胞骨架功能、醣類代謝功能、抗氧化還原活性功能、訊息傳遞功能與運輸蛋白質功能。酸性纖維母細胞成長因子(aFGF)可能提供一個有利於神經修復的環境，透過細胞內相關蛋白質協力合作來降低慢性期之神經傷害。本篇研究除了在學術層面上更深入提供對於脊髓損傷大鼠在晚期神經再生機制之觀點外，希望在未來可以發展出新的脊髓損傷治療策略，進而對於脊髓損傷病人臨床治療應用上有所貢獻。

Acidic fibroblast growth factor (aFGF), acts as a potent neurotrophic factor, could stimulate the survival of neurons and re-growth of neurite in the injured spinal cord area. However, the molecular mechanism of protective effect of aFGF in vivo is still not clearly or fully understood. Previous studies showed that significant improvement in the locomotor behavior analysis resulted from of the aFGF-treated rats after contusive spinal cord injury (SCI). SCI will initiate the different pathological changes along with time, it is therefore, we would like to propose that aFGF may play different role at different time course during the repair processes. Proteomic and bioinformatic approach were adapted to investigate the protein profile changes of the damaged spinal cord tissue of the SCI rats treated either with or without aFGF at 28 days after contusive injury. We found that 25 protein spots were shown to have significant differential expression using proteomic approach. Using bioinformatic analysis, these proteins were categorized into five major expression patterns and five functional categories including structural activity, glucose metabolism, antioxidant/oxidoreductase activity, signal transduction and transporter activity according the biological process in which they are involved. Hence, the protective effect of aFGF at the late stage came from a set of teamwork of multiple factors that may attenuate neuronal injuries for providing a better environment recovering from the spinal cord damage. In this study, our results provide a better insight into the SCI regeneration mechanisms in the late stage of repair processes of SCI rat. Noticeably, in addition to shed light on the academic contribution to the mechanism of neural regeneration, the outcome of this research will also be beneficial for the clinical application of the SCI patients.

中文摘要....................................................................................................1
英文摘要....................................................................................................2
第一章、緒論.............................................................................................4
一、研究動機背景.................................................................................4
二、研究目的...........................................................................................7
第二章、文獻回顧...................................................................................................8
一、脊髓損傷簡介.............................................................................................. 8
二、脊髓神經損傷之病理...............................................................................10
三、抑制脊髓神經再生之分子機制............................................................11
四、脊髓神經修復的策略.............................................................................. 13
五、酸性纖維母細胞成長因子 (aFGF) ................................................... 14
第三章、實驗方法…………………......................................................................18
一、實驗動物......................................................................................................18
二、動物脊髓損傷手術.................................................................................18
三、aFGF的注射實驗.....................................................................................19
四、行為測試及評估........................................................................................19
(一)綜合行為評分表( CBS Score )………………………………………….. 19
(二)開放場域行走測試評分表(BBB Score)…………………………… 20
五、統計分析....................................................................................................20
六、蛋白質樣品的萃取.................................................................................21
七、蛋白質濃度的測定.................................................................................21
八、SDS-PAGE蛋白質電泳分析................................................................22
膠體製作及電泳......................................................................................22
九、二維蛋白質電泳分析…………………………………………...………23
(一)第一維等電點聚焦實驗..........................................................................23
(二)第二維SDS-PAGE分析……………………………………...…………24
十、二維蛋白質電泳染色...............................................................................25
(一)Coomassie blue 染色...............................................................................25
(二)Silver stain 染色...............................................................................25
十一、二維蛋白質電泳軟體比對分析.......................................................26
十二、利用質譜儀進行蛋白質身分鑑定...................................................26
十三、生物資訊學分析方法..........................................................................27
第四章、結果.........................................................................................................28
一、利用蛋白質體策略分析鑑定正常組、損傷組和加藥組間
其蛋白質表現圖譜差異之結果………………..………………........28
二、蛋白質表現型態與階層式分群法分析結果…………….…………29
三、蛋白質生物性功能分類結果………………………….………………31
第五章、討論…………………………………………………...…………………33
一、 酸性纖維母細胞成長因子(aFGF)對氧化性壓力
(oxidative stress)之探討......................................................................34
二、酸性纖維母細胞成長因子(aFGF)對生化代謝功能
之探討………………………………………………………………….…37
三、酸性纖維母細胞成長因子(aFGF)對細胞骨架功能
之探討…………………………..………………………...........................37
四、酸性纖維母細胞成長因子(aFGF)對調控星狀細胞(astrocyte)
之探討 ......................................................................................................39
五、酸性纖維母細胞成長因子(aFGF)對蛋白穩定度
之探討.........................................................................................................41
六、 總結.............................................................................................................41
第六章、參考文獻..................................................................................................44

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