近年來，許多臨床試驗證實間葉幹細胞 (mesenchymal stem cell, MSC) 細胞療法應用於許多種疾病中，是具有展望性的治療方法，但移植後的低細胞存活率限制了其療效。現存可提升細胞存活率的方法，皆仍存有各自的應用限制。一氧化氮為生物體內重要的訊息傳遞者，已被證實可提升細胞抗氧化能力，具細胞保護效果。另有文獻指出其會影響 MSC 多種調節能力，其中以促血管新生能力最被廣為探討，但根據一氧化氮供體與使用濃度不同，細胞表現出截然不同的結果，至於免疫調節能力則目前仍無文獻探討。比起其他一氧化氮供體，雙亞硝基鐵錯合物 (dinitrosyl iron complex, DNIC) 具有許多優勢，然而仍無文獻探討其對於 MSC 的影響。在本篇研究中，我們利用 DNIC 刺激 MSC 的方式，一為提升移植後的細胞存活率，二是探討其對於MSC多種調節能力之影響。結果顯示，比起市售一氧化氮供體S-Nitroso-N-acetyl-DL-penicillamine，DNIC 的一氧化氮釋放曲線可更有效提升細胞的第一型血紅素氧化酶基因與蛋白質表現，於氧化壓力下展現良好細胞保護效果。由促血管新生相關基因和蛋白質表現變化，證實在 DNIC 刺激後不會抑制細胞的促進血管新生能力。由多樣性磁珠偵測系統分析結果可知，DNIC 會影響細胞所分泌的細胞激素，尤其是 interleukin-1 receptor antagonist (IL-1RA)，並且其條件培養基可抑制微膠細胞往 M1 極化和促發炎因子的表現。另外，經 DNIC 刺激的 MSC 於發炎環境下會顯著提升 IL-1RA 基因表現，因此本研究認為 DNIC 的作用可能是透過提高細胞 IL-1RA 基因與蛋白質表現，使其具更好的免疫抑制能力。在動物實驗中，將細胞植入小鼠腦中風的受損區域後，利用非侵入式 3D 活體分子影像系統追蹤植入細胞存活情況。結果顯示移植後短時間內，有 DNIC 刺激過的細胞確實具較高細胞存活率。綜論以上結果，利用 DNIC 刺激 MSC 的方法，可提升移植後細胞存活率；更重要的是該方法不但不會抑制細胞促血管新生能力，還能提升其免疫調節潛能。未來作為提升 MSC 移植後存活率及治療潛能的方法，是具有展望性的。

Over the past decades, mesenchymal stem cell (MSC)-based therapy has been shown great therapeutic potential in many different pathologies. However, the poor survival rate after transplantation is a crucial reason accounting for hampering the repair effect of MSC. To date, some strategies have been used to solve this problem, but there are still some obstacles that need to be overcome. Nitric oxide is an important messenger that mediates diverse biological functions. Some researchers have suggested that nitric oxide could exert a cytoprotective effect and regulate multiple functions on MSC. However, the half-life of nitric oxide is extremely short, therefore series of nitric oxide donors have been developed. Dinitrosyl Iron Complex (DNIC) is a nitric oxide donor with lots of advantages over others, but it has never been reported for its functions on MSC. We therefore pre-actived MSC with DNIC to evaluated their cytoprotective effect on MSC and the influence on MSC’s pro-angiogenic and immunomodulatory potential. The results of in vitro study revealed that compared with another nitric oxide donor, S-Nitroso-N-acetyl-DL-penicillamine, DNIC with a specific release profile could more significantly elevate heme oxygenase-1 mRNA and protein expression in MSC, thus further improving cell viability under oxidative stress. The results of pro-angiogenic related gene and protein expression exhibited that DNIC does not suppress MSC’s pro-angiogenic ability. Besides, it could promote MSC’s immunosuppressive capacity, and further inhibits microglia M1 polarization and pro-inflammatory cytokines expression, which may resulted from increasing IL1RN/IL-1RA expression. The results of in vivo study demonstrated that MSC primed with DNIC indeed showed better cell viability in short-term period after transplanting cells into ischemic stroke mice. To sum up, MSC primed with DNIC is very promising as a strategy for improving post-engrafted survival and enhancing therapeutic potential.

摘要...............................................................I
Abstract..........................................................II
誌謝..............................................................III
目錄..............................................................IV
圖目錄...........................................................VIII
表目錄.............................................................XI
第一章、緒論........................................................1
1-1、間葉幹細胞.....................................................1
1-2、間葉幹細胞療法 .................................................3
1-2-1、促進血管新生能力..............................................3
1-2-2、促神經再生能力................................................4
1-2-3、免疫調節能力..................................................5
1-3、細胞療法的主要限制.............................................11
1-4、提升間葉幹細胞存活率之方法......................................12
1-4-1、與生醫材料共移植系統.........................................12
1-4-2、與其他細胞共移植.............................................14
1-4-3、基因工程 (gene modification)................................14
1-4-4、預處理細胞(Pre-conditioning)................................15
1-5、一氧化氮對於幹細胞之影響.......................................18
1-5-1、 細胞存活、增生及遷移能力....................................19
1-5-2、血管新生能力................................................22
1-5-3、分化能力...................................................26
1-5-4、免疫調節能力................................................27
1-6、一氧化氮供體..................................................28
1-7、研究動機與實驗目的............................................32
第二章、材料和方法.................................................35
2-1、格里斯試驗 (Griess assay).....................................35
2-2、細胞培養......................................................36
2-3、一氧化氮供體配置及預處理細胞....................................37
2-4、配置瓦解之雙亞硝基鐵錯合物(decayed DNIC)........................37
2-5、細胞存活率試驗 (Cell viability test)...........................37
2-6、細胞存活/死亡染色 (Live/Dead Staining).........................38
2-7、抗氧化壓力試驗.................................................38
2-8、條件培養基(conditioned medium)製備.............................38
2-9、體外發炎模型建立...............................................39
2-10、間葉幹細胞與微膠細胞共培養系統(Transwell co-culture system)....39
2-11、即時聚合酶連鎖反應(Quantitative real-time PCR)................40
2-12、西方墨點法 (Western blotting)................................46
2-13、酵素連結免疫吸附分析法(ELISA).................................50
2-14、多樣性磁珠偵測系統 (Bio-Plex® Multiplex Assays)...............50
2-15、小鼠腦中風動物模型建立(middle cerebral artery occlusion, MCAO)52
2-16、統計方法.....................................................53
第三章、實驗結果與討論..............................................54
3-1、一氧化氮供體之釋放曲線.........................................54
3-2、一氧化氮供體對於細胞存活率之影響................................55
3-2-1、不同一氧化氮供體濃度下的細胞存活情況...........................55
3-2-2、促細胞存活/凋亡相關基因表現 ..................................57
3-3、一氧化氮供體對細胞型態之影響....................................58
3-4、一氧化氮供體調節第一型血紅素氧化酶表現及可能機制探討..............59
3-4-1、HMOX1 基因表現..............................................59
3-4-2、Heme oxygenase-1蛋白質表現..................................61
3-4-3、雙亞硝基鐵錯合物提升HO-1的可能機制之探討.......................62
3-5、一氧化氮供體於氧化壓力下對細胞保護之能力.........................65
3-6、雙亞硝基鐵錯合物對於細胞的促進血管新生潛能之影響..................67
3-7、雙亞硝基鐵錯合物對於細胞的免疫調節能力之影響.....................68
3-7-1、正常培養環境下對免疫調節相關基因與蛋白質之影響..................68
3-7-2、條件培養基對微膠細胞之影響....................................71
3-7-3、發炎環境下對免疫調節基因之影響................................72
3-7-4、共培養系統對於間葉幹細胞的免疫調節基因之影響...................74
3-8、動物實驗......................................................75
第四章、結論.......................................................78
參考文獻...........................................................79

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