系統化分析microRNA於不同來源間葉幹細胞中成骨與脂肪分化能力歧異度之調控作用

Abstract

計畫名稱：系統化分析microRNA 於不同來源間葉幹細胞中成骨與脂肪分化能力歧異度 之調控作用 成體幹細胞的因具有易取得且不具有侵入性等優點已成為重要的研究標的，其中間葉 幹細胞 (mesenchymal stem cells, MSCs) 在mesenchyme-lineage 的分化特性與能力已成 為相關研究不可獲缺的研究模式。目前已瞭解在間葉細胞的分化途徑中，成骨細胞分化 與脂肪細胞分化呈現反向作用，當脂肪細胞分化能力越強時，成骨細胞分化能力則隨之 減弱。由於MSCs 為成骨細胞與脂肪細胞的前趨細胞，因此以MSCs 分析二分化之間的 歧異點是非常重要的。由於不同來源的MSCs 具有不同mesenchyme-lineage 的分化能 力，造成這種差異的原因目前仍未知，相關研究顯示幹細胞通常藉由抑制多種不同基因 組使細胞進入早期分化，顯示micro RNA (miRNA)的調控對於幹細胞所具有的原始分化 能力極具重要性。 我們先前研究發現骨髓MSCs 與臍帶血MSCs 具有不同mesenchyme-lineage 分化能 力，臍帶血MSCs 擁有較強的成骨細胞分化能力但其分化為脂肪細胞的能力卻十分低， 而骨髓MSCs 則具有較強的脂肪分化能力，相對地其成骨細胞分化能力卻弱於臍帶血。 造成這種差異的原因目前仍未知，本計畫中我們藉由瞭解miRNA 在骨髓MSCs 與臍帶 血MSCs 中的表現情形，並分析miRNA 對於早期調控成骨細胞分化與脂肪細胞分化途 徑的關連性，以獲得miRNA 對MSCs 具有不同分化能力的可能調控機制。我們以骨髓 MSCs 與臍帶血MSCs 作為研究平台，目前已完成未分化時二者基因的表現分析。我們 發現在未分化狀態下，二MSCs 即表現出產生分化能力歧異的基因。因此本計畫第一年 我們將利用骨髓MSCs 與臍帶血MSCs 其cDNA array 與miRNA array 所得結果，以生物 資訊方式預測出調控二MSCs 中具表現差異基因之miRNA。再藉由功能分析，了解骨 髓MSCs 與臍帶血MSCs 中miRNA 的調控目標，系統化地尋找可能造成分化能力差異 的miRNA 標的。第二年我們將以第一年所分析出之結果為基礎，利用miRNA knockdown 與over-expression 技術，進行MSCs 成骨細胞分化與脂肪細胞分化的觀察，並分析與分 化相關之miRNA 與專一基因的表現狀況。最後將以羊水MSCs 與脂肪組織MSCs，於 未分化時偵測此群miRNA 表現結果，並觀察其成骨細胞分化與脂肪細胞分化能力之差 異，以確認我們所獲得的結論。 我們相信越瞭解每一種MSCs 特性，對未來進行細胞治療時選擇來源細胞可更為精 確。本計畫的執行可瞭解miRNA 對於不同來源MSCs 分化能力歧異的重要性，且可確 認成骨作用與脂肪細胞形成作用相互關係中miRNA 所扮演的調控機制。由於目前認為 成骨細胞分化與脂肪細胞分化的反向分化能力為骨質疏鬆症的成因之一，我們相信本計 畫的研究成果亦可提供骨質疏鬆症與脂肪形成關係間，miRNA 所參與的調控作用。

Systematic analysis reveals the roles for microRNA in adipogenesis and osteogenesis of human mesenchymal stem cells Mesenchymal stem cells (MSCs) have been known as the differentiated model for mesenchyme-lineages. An inverse relationship between the differentiation of adipocytes and osteoblasts was demonstrated in cell and animal models and found in various human diseases, including osteoporosis. Because osteoblasts and adipocytes arise from the same MSCs precursors, the osteogenesis and adipogenesis could be “switched” during human body. The reciprocal relationship between adipogenesis and osteogenesis was demonstrated; however, the mechanisms remain largely unknown. Recently, microRNA (miRNA) modulates gene expression by inhibiting the translation or by promoting the degradation of target mRNAs. Several studies indicated that miRNA act as key regulators in cell differentiation. MSCs isolated from different sources have been demonstrated to have various differentiated potentials. In previous study, we observed that MSCs had various differentiated potentials from bone marrow and umbilical cord blood. Cord blood MSCs have a significantly stronger osteogenic potential but less capacity in adipogenic differentiation than bone marrow MSCs. We have compared the gene expressions between cord blood and bone marrow MSCs by cDNA array chip. Several known genes which involved in osteogenesis and adipogenesis were found from cDNA chip results. Recently, some evidences show that adipogenesis and osteogenesis could be regulated by miRNA. In this study, we try to understand the roles of miRNA in osteogenesis and adipogenesis between MSCs-derived from different sources . In this study, we will use the various genes from our cDNA chip as regulation targets, and the functional miRNAs will be prediction in silico. These prediction results will be confirmed by miRNA chip analysis. Following by functional analysis, the miRNAs which involved in early osteogenesis or adipogenesis will be selected as study targets. For further understanding the roles of the selected miRNAs in osteogenesis and adipogenesis, the analysis were accomplished by miRNA knockdown and over-expression tests in bone marrow MSCs and cord blood MSCs. Finally, the expression of the targeted miRNA will be observed in MSCs -derived from amniotic fluid and –derived from adipose tissue to confirm our findings. In this study, we will systematic compare the differences of miRNA expression between bone marrow- and cord blood-derived MSCs, and try to understand how miRNA regulate the diverse potentials. We believe that our results will not only provide the connections for miRNA regulation in MSCs differentiation but also provide new information for further therapeutic applications.