2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660560摘要：影響停經後婦女非常嚴重的骨質疏鬆症(Osteoporosis)，主要是由於成骨細胞(Osteoblast) 在代謝過程中產生相對不足所致。而成骨細胞的前驅細胞── 間質幹細胞(Mesenchymal Stem Cell, MSC)，它具有多重分化的潛能，可以在轉錄因子RUNX2,PPARγ2, SOX9 及各自的協同調節因子(co-modulators) 的共同作用下，分別分化為成骨細胞、脂肪細胞及軟骨細胞。自然界中當細胞受到外界的壓力或老化時，會產生活性氧化物(Reactive oxygenspecies－ROS)來參與細胞的各種生理作用。我們在MSC 的研究初步顯示：ROS 會抑制MSC 往成骨細胞方向的分化，而增加往脂肪細胞方向的分化，但其確切的調控角色及作用機轉仍不清楚。另外軟骨細胞的分化，和成骨細胞、脂肪細胞的分化方向之間，是否亦有相互影響，目前也都還未被深入探討。我們實驗室之前一直從事植物性荷爾蒙(phytoestrogen)、Osteoblast 及MSC 的相關研究(Stem Cells 2007；Stem Cells 2009； J Biol Chem 2010；Biomaterials in press等)。目前文獻上所知RUNX2 及PPARγ2 的協同調節因子，而與ROS 有關的，包括有抗老化基因SIRT1 及抗壓力基因FOXO3A 等。我們之前發現植物性荷爾蒙白黎蘆醇(resveratrol)可以同時增加骨密度及抑制乳癌細胞，其機轉分別是透過FOXA1 及FOXO3A (發表在J Biol Chem 2007)。而FOXO 在幹細胞的分化及凋亡，已被學者報告占有極重要的角色(Cell 2007)。值得注意的是，白黎蘆醇也是抗老化基因SIRT1 的興奮劑(agonist)，而SIRT1 已被報告可抑制脂肪細胞中PPARγ 的活性。我們最近也發表，SIRT1 可以透過和FOXO3A 結合，在MSC 中來活化RUNX2 基因的轉錄和表現(J BoneMiner Res 2011)。因此我們想要進一步來探討：氧化壓力(活性氧化物)對MSC 分化方向的影響及其作用機轉。希望藉由誘導或抑制細胞內ROS 的產生，來探討當MSC 走向特定分化方向時，SIRT1/FOXO3A 和三個主要的轉錄因子PPARγ2、RUNX2 以及SOX9 之間的分子交互調控作用。吾人計劃朝如下的目標去進行目標一:評估並比較ROS 對不同組織來源的MSC 分化方向之影響（第1-6 個月）理由：之前的觀察發現，不同組織的MSC 分化成下游細胞的方向及能力並不一樣。由於我們的初步研究證實ROS 可以影響MSC 的分化方向，因此我們想進一步瞭解，不同組織來源的MSC，其本身存在的內源性ROS 濃度，是否會影響其分化的方向及能力。目標二:進一步探討ROS 調控SIRT1/FOXO3A 和主要轉錄因子(PPARγ2、RUNX2、SOX9) 活性的分子交互調控機制（第7-24 個月）理由：由我們初步的研究發現，ROS 可以影響SIRT1 及FOXO3A 的表現。而SIRT1 是PPARγ2 及RUNX2 在轉錄作用時共同的co-modulator，FOXO3A 也具有抗氧化的作用。因此我們想來進一步釐清，在MSC 往不同的方向分化時，其中彼此之間的分子調控機轉。目標三: 在活體動物實驗中，驗證ROS 對MSC 多功能分化潛能的影響（第25-36 個月）理由：為了驗證前面實驗室中所得到的結果，我們將長期餵食小鼠酒精（慢性氧化壓力的動物模式），同時給予或不給予ROS 清除劑，觀察之後其MSC 分化方向與能力的變化。本計劃的研究成果，將能使吾人能釐清氧化壓力對間質幹細胞分化方向的影響，以進一步奠定骨質疏鬆症等重要老化疾病治療的基礎。<br> Abstract: Postmenopausal osteoporosis, a disease of increasing prevalence, is due to insufficientosteogenesis by osteoblasts, which is an important differentiated cell type of mesenchymalstem cells (MSCs). Lineage-specific differentiation of MSCs into adipocytes, osteoblasts andchondrocytes is transcriptionally initiated by the master transcription factors (TFs) PPARγ2,RUNX2, and SOX9, respectively.Our lab has been interested finding new sources of MSCs (Yen et al., 2007, Yen et al.,2011) and lineage commitment mechanisms — especially osteogenesis (Su et al., 2007; Suet al., 2010; Tseng et al., 2011a) — of these versatile adult stem cells (Tseng et al., 2011b).Recent studies have revealed that the actions of these master lineage-specific TFs are furtherfine-tuned by co-modulators and other small molecules. Reactive oxygen species (ROS),created by cellular stresses, are known to regulate various physiological functions andpathological diseases. Recently, ROS has been shown to facilitate adipogenesis and inhibitosteogenesis in preadipocytes—progenitors of adipocytes—and MSCs. However, little isknown about how the intrinsic redox states of MSCs affect differentiation capacity. Moreover,the detailed molecular mechanism of ROS on directing lineage commitment of MSCs is stillunknown.There are transcriptional co-modulators of MSC master lineage-specific TFs whichalso interact with ROS, and these include SIRT1—a histone deacetylase with a role inlongevity— and FOXO3A, a stress-resistant gene. An important agonist of SIRT1 isresveratrol, and our lab has previously reported on the protective effects of thisnaturally occurring polyphenol on a number of cell types including osteoblasts (Lin et al.,2003; Yen et al., 2005; Su et al., 2007). Most recently, we found that resveratrol canenhance MSC osteogenesis through cooperation with FOXO3A to activate RUNX2 genetranscription and downstream osteogenic program in human embryonic stem cell-derivedmesenchymal progenitors (EMPs) (Tseng et al., 2011a) — a source of renewable MSCs whichwe have developed a robust method of isolation (Yen et al., 2011).We therefore propose to investigate how ROS regulates lineage commitment ofMSCs by studying effects of H2O2 on the molecular interactions between the master TFsPPARγ2 and RUNX2, and the co-modulators SIRT1 and FOXO3A. Our Aims will be to (1)evaluate the effects of ROS on lineage commitment of MSCs isolated from distinctsources, (2) explore the regulatory mechanism of ROS on the transcriptional activities ofPPARγ2 and RUNX2 and interactions with SIRT1/FOXO3A, and (3) examine the in vivorole of ROS on the multipotency of MSCs.We anticipate that our Proposal will elucidate the transcriptional crosstalk betweenPPARγ2 and RUNX2, and role of SIRT1/FOXO3A in fine-tuning stem cell lineage switch, aswell as provide a molecular explanation for oxidative stress-directed lineage commitment ofMSCs.間質幹細胞分化方向氧化壓力活性氧化物mesenchymal stem cellslineage commitmentoxidative stressreactive oxygen species (ROS)