利用肌肉前驅細胞來探討體細胞多能性誘發的機制; Using Myogenic Precursor Cells to Elucidate the Mechanisms of Induced Pluripotency of Somatic Cells

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題名:	利用肌肉前驅細胞來探討體細胞多能性誘發的機制;Using Myogenic Precursor Cells to Elucidate the Mechanisms of Induced Pluripotency of Somatic Cells
作者:	陳盛良
貢獻者:	生命科學系
關鍵詞:	Oct4;Nanog;iPS;pluripotency;幹細胞;肌肉;Oct4;Nanog;iPS;pluripotency;muscle;stem cell;生物技術
日期:	2010-08-01
上傳時間:	2011-07-13 16:17:15 (UTC+8)
出版者:	行政院國家科學委員會
摘要:	對傳統治療方式無法治癒之基因損壞所導致的遺傳性疾病而言，藉由移植幹細胞至受損部位進行修補，並輔以傳統治療，可能是唯一目前有希望可以達到良好治療效果的方法。但胚胎幹細胞來源有限，且其數量及種類稀少，甚至移植於人體內需考量免疫排斥作用的干擾。為避免免疫排斥反應生成，可使用自體幹細胞來進行治療。但自體幹細胞不易從病人體內分離以及大量培養，如果可選擇使用數量較多且易取得之體細胞作為替代，以體外培養的方式，誘導其回復至前驅細胞型態，則可同時解決免疫排斥以及細胞來源的問題。近年的研究指出，於小鼠和人類纖維組織母細胞中大量表現 iPS factors (Oct4、Sox2、Klf4 及 c-Myc) 能誘發其形成 induce pluripotent stem cells (iPS cells)，然而 c-Myc 及 Klf4 為致癌基因，會導致腫瘤形成，若應用於醫療行為，具有相當的潛在危險性，因此必須避免使用。目前已有研究顯示，使用 Oct4、Sox2 及 histone deacetylase 抑制劑 valproic acid 即能成功誘使人類纖維組織母細胞形成 iPS cells。骨骼肌佔人體重量40％，內含有大量稱為satellite cell 的幹細胞，因此具有高可塑性及再生能力，且易自病人體內分離培養，所以 satellite cell 可作為自體幹細胞的最佳來源之一。直至今日，許多轉殖技術已應用於使細胞大量表現 iPS factors，包括 retroviral transduction 、adenoviral transduction 及plasmid transfection 等，其中 retroviral transduction 具有較高轉殖效率，但缺點為容易產生 integration mutagenesis 的情形，因此迫切需要找尋能有效增加 iPS factors 表現量及提升病人治療安全性之方法，因此本計劃的目的是希望能藉由誘發肌肉前驅細胞的多能性，來了解iPS factors 誘發體細胞多能性的機制，同時藉由篩選更有效的小分子化合物以及重組 iPS factos 蛋白來建立一更安全有效率的細胞重建 (reprogramming)技術。未來希望能應用於再生醫學治療，提供因基因缺損而導致的遺傳性疾病另一有效治療方法。本計畫著重於四個主要目標為： (1) 藉由大量表現 iPS cell factors 於肌肉前驅細胞，來了解iPS factors 誘發體細胞多能性的機制。 (2) 篩選出促進細胞多能性之小分子。 (3) 建立攜帶由Oct4 啟動子驅動的GFPneo 報導基因的轉殖鼠。 (4) 利用小分子化合物以及重組iPS factos 蛋白來建立一更安全有效率的細胞重建 (reprogramming)技術 A great hurdle to cell mediated tissue regeneration therapy is the possible immune rejection of the graft by the host. Therefore, adopting autologous stem cells in this therapy becomes very critical to its success. Unfortunately, stem cells from the patient’s body are difficult to isolated and expand ex vivo. However, if the patient’s somatic cells, which are in large amount and easy to get, can be reprogrammed and induced to trans-differentiate into precursors of target cell types in vitro, then both the cell number and immune rejected problems can be solved. In recent years, several groups have found that over-expression of Oct4 and Sox2 together with either Klf4 and c-Myc or Nanog and Lin 28 can induce pluripotent stem (iPS ) cells from mouse and human fibroblasts with germline-competency. One of the studies has successfully induced iPS cells from human fibroblasts by using only Oct4 and Sox2 supplemented with a histone deacetylase inhibitor, valproic acid (VPA), although the efficiency is low (0.001%) as compared to that of using 4 factors (0.1%). These studies strongly demonstrated that reprogramming somatic cells to stem cells is feasible and it can be achieved either by simply introducing 2-4 stem cell-specific factors into somatic cells or in conjunction with pluripotency-promoting compounds, such as VPA. To date, several methods, including retroviral and adenoviral transduction and plasmid transfection, have been employed to over-express these iPS factors Both retroviral transduction and plasmid transfection raised the concern of insertion mutagenesis and introduction of ecotopic DNA. Adenoviral transduction avoids the insertion mutagenesis, but unfortunately, the efficiency achieved by this approach is relatively low. Therefore, new approaches need to be taken to avoid the problem of insertion mutagenesis and in the mean time increase the iPS induction efficiency. Skeletal muscle constitutes about 40% of adult body weight and is one of the most accessible tissues in a patient’s body. It is highly plastic and can regenerate readily upon physical and chemical damages due to the existence of large number of stem cells in this tissue. Taking advantage of its large amount of stem cells and easy accessibility, skeletal muscle stem cells can serve as a reliable source of autologous stem cells for reprogramming. Therefore the specific aims of this study are: 1. Reprogramming myogenic precursor cells by (vector-dependent) over-expressing iPS factors and analyzing their expression profile 2. Screening of pluripotency promoting small compounds 3. Generating transgenic mice carrying Oct4 promoter driven GFPneo reporter 4. Establishing a vector-free reprogramming procedure 研究期間：9908 ~ 10007
關聯:	財團法人國家實驗研究院科技政策研究與資訊中心
顯示於類別:	[生命科學系] 研究計畫

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