2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644209摘要：更年期後的骨質疏鬆症因人口的老化變得愈來愈常見，而因骨質疏鬆症所導致的骨折，往往會造成病人很大的痛苦及家庭社會的負擔。我們實驗室一直以來即在從事骨質疏鬆症治療方法的研究，其中包括植物性荷爾蒙，如白藜蘆醇(Resveratrol)，在成骨細胞及其前驅細胞－間葉幹細胞的骨化作用(osteogenesis)機轉，所獲致的研究成果分別已刊登在2007 年的J Biol Chem (迄今被引用57 次)及2011 年的J Bone Mineral Res(迄今被引用41 次)。間葉幹細胞因其取得較容易，且沒有胚胎幹細胞的倫理爭議及形成腫瘤的疑慮，目前在細胞治療的研究上已漸成主流。但因其細胞數量少，如欲應用到臨床常需大量繼代增殖，而這過程卻往往也伴隨著細胞的老化，甚至影響其之後分化的方向，如減少骨化的能力等。因此，闡明間葉幹細胞老化的機轉是其在臨床應用前重要的課題。我們實驗室最近發表的論文顯示：間葉幹細胞在經歷多次的繼代增殖後，會有氧化反應物(ROS)的積聚，進而造成它的老化及分化方向的改變(減少往成骨細胞分化）。有趣的是，我們接下來的實驗初步顯示，這些不利的現象似乎都是受到c-MAF 這個分子的調控。而由文獻知道c-MAF 除了是ROS-sensitive 外，也是骨化時主要轉譯因子RUNX 2 的協同因子。雖然以前大家就知道植物性荷爾蒙有很強的抗氧化能力，但關於它在減少間葉幹細胞老化及增進骨化能力這些方面的作用機轉，則尚未釐清。因此，我們希望提出下列為期三年的研究計劃，來探討氧化壓力及c-MAF 調控人類間葉幹細胞老化及骨化能力的作用機轉，進而在動物實驗篩選驗證植物性荷爾蒙對骨質疏鬆症的療效。我們的研究目標包括：目標一：篩選出能減少間葉幹細胞老化及增進骨化的植物性荷爾蒙。方法主要是在實驗室中測試各種植物性荷爾蒙，看哪些最能減少間葉幹細胞ROS 的積聚及老化，並觀察其往成骨細胞或脂肪細胞分化方向改變的差異。且比較所篩選出來的植物性荷爾蒙在加入老化間葉幹細胞後其轉譯體前後的變化目標二：闡明在目標一中所篩選出來的植物性荷爾蒙，其與c-MAF 及RUNX 2 之間相互的分子調控機轉。方法主要是要評估所選定的植物性荷爾蒙，對骨化、脂肪化及老化等調控基因群組表現的影響，及參照目標一轉譯體變化的結果，用分生的方法來探討c-MAF 與RUNX 2 在其中的分子調控機轉。目標三：在動物活體驗證所篩選出植物性荷爾蒙的抗氧化／抗老化及促骨化的效果。我們希望經由這個研究的進行，能讓我們進一步釐清間葉幹細胞骨化的作用機轉，進而提供一個可以篩選具療效植物性荷爾蒙或其它小分子的平台，以做為臨床治療骨質疏鬆症之用。<br> Abstract: Postmenopausal osteoporosis is an increasingly prevalent disease due to worldwide aging populations.Characterized by decreased bone mass resulting in bone fragility, this silent disease greatly increases the riskof bone fractures and leads to profound morbidity and mortality. Our research interests and goal has beento evaluate therapeutic compounds—including phytoestrogens such as resveratrol (RSV)—forosteoporosis by investigating molecular mechanisms involved in osteogenesis at the levels of theosteoblast—the cell type responsible for bone formation—and its progenitor, the mesenchymal stemcell (MSC). ( J Biol Chem 2007, cited 57 times; J Bone Miner Res 2011, cited 41 times )MSCs are multipotent somatic stem cells without ethical problem, which are excellent candidates fornumerous therapeutic applications. However, the vigorous ex vivo expansion of MSCs to reach the cellnumbers necessary for clinical application usually result in senescence, with detrimental effects onself-renewal/proliferation and differentiation capacity, diminishing osteogenic potential in particular.Therefore, elucidation of the mechanisms involved in MSC senescence is critical to its clinical application.Our recent published work has demonstrated that prolonged in vitro expansion of adult MSCsresults in the accumulation of reactive oxygen species (ROS) which are involved in decreasingproliferative potential and multilineage differentiation capacity especially osteogenesis. ( Antioxidant RedoxSignal 2013, impact factor=7.6) Interestingly, our current data demonstrate that many of thesedetrimental effects converge on one factor: c-MAF, an ROS-sensitive transcription factor which is alsoa co-factor of Runx2, the master lineage transcription factor for osteogenesis. It is well known thatmany phytoestrogens are effective antioxidants, but studies are mainly correlative without cleardemonstration of mechanisms of action; moreover, additional connections to osteogenesis are not wellelucidated.Based on our experience and continued interest, we would like to explore in this 3-year Proposal, themolecular role of oxidative stress and c-MAF in mediating senescence and osteogenic capacity ofhuman mesenchymal stem cells (MSCs), and the use of phytoestrogens as therapeutic agents in these 2capacities. Our Aims in this Proposal will be to :Aim 1: Screen for phytoestrogens which can reduce MSC senescence and enhance osteogenesisa. Assay for candidate phytoestrogen which reduce ROS and subsequent senescent phenotypic changein MSCsb. Assay for candidate phytoestrogens which induce osteogenic differentiation and inhibit adipogenicdifferentiation in MSCsc. Perform transcriptome analysis on senescent MSCs +/- phytoestrogen treatment (selected fromabove assays) and young MSCsAim 2: Elucidate the mechanisms involved in Aim 1 results with regards to c-MAF and Runx2a. Assess candidate phytoestrogens for ability to modulate gene expression profiles involved inosteogenesis, adipogenesis, and senescenceb. Elucidate whether candidate phytoestrogens exert transcriptional control of c-MAF and Runx2(PPARγ also if necessary) by assessing promoter activities of these genes, protein expression levels,and modulation of downstream genesc. Investigate pathways highlighted by novel transcripts identified from Aim 1.c transcriptome analysisAim 3: Validate in vivo the anti-senescence/antioxidant and pro-osteogenesis effects of selectedphytoestrogensWe hope our work in this Proposal will allow for further mechanistic understanding of the mechanisms ofosteogenesis, as well as provide an efficient platform for selection of candidate phytoestrogens and likelyother small molecules for therapeutic application on osteoporosis.骨質疏鬆症植物性荷爾蒙間葉幹細胞氧化壓力氧化反應物osteoporosisphytoestrogenmesenchymal stem cellsoxidative stressreactive oxygen species (ROS)