人類骨髓及脂肪間質幹細胞功能性基因體比對分析
Functional genomic characterization of mesenchymal stem cells isolated from human bone marrow and adipose tissues
中文摘要
人類間質幹細胞最初是由成人骨髓中所分離而得。在近年來的研究中，科學家從 成人其他組織也成功分離具有骨髓間質幹細胞特性的細胞族群。間質幹細胞在人 體中藉由分泌許多細胞生長因子，維持組織正常功能與再生修復。並且藉由提供 細胞外基質成分、分泌驅化因子，提供人體其他幹細胞/前驅細胞族群微環境並 且驅回該組織進行修復的正常機制。過去的研究發現由不同組織分離出的間質幹 細胞表現有相似的細胞表面分子，以及多重分化為中胚層結締組織細胞類型的能 力。然而對於不同組織來源的間質幹細胞特性以及功能差異卻很少被研究探討。
為了更進一步了解組織間質幹細胞分子層面上的差異，本實驗利用人類互補去氧 核糖核酸微矩陣列分析以及半定量反轉錄聚合酶連鎖反應，就脂肪組織間質幹細 胞與骨髓間質幹細胞，從事基因體表現比較分析。就多重分化路徑相關基因以及 胚胎早期基因的表現比較，我們發現兩者在這些基因表現上的程度相近，反應脂 肪間質幹細胞與骨髓間質幹細胞具有類同之細胞原始性及分化潛能。這樣的結果 與體外分化培養所觀察到的結果符合。我們更進一步由晶片實驗的結果蒐集各別 高度差異表現的基因作比對分析得知，這些表達差異基因主要包含生長因子、細 胞激素、細胞表面分子、細胞外基質分子、以及受器，顯示脂肪間質幹細胞與骨 髓間質幹細胞主要的特性差異可能在於細胞與細胞之間、細胞與微環境之間的調 控功能。本實驗確認數個細胞激素/生長因子基因在脂肪間質幹細胞與骨髓間質 幹細胞有許多顯著差異的基因表現：骨髓間質幹細胞在 LIF, SDF-1, BMP-4, PlGF-1, PlGF-2, HGF, HB-EGF, Jagged-1 基因表現明顯高於脂肪間質幹細胞。也 初步利用 Western blottong 確認 LIF, SDF-1, HGF 在蛋白質表現量上的差異。說明 了這些差異因子可能造成骨髓以及脂肪來源的間質幹細胞在調控其他幹原/前驅 細胞回歸機制以及對於造血以及血管新生過程的輔助能力上有所差異。此外，骨 髓間質幹細胞以及脂肪間質幹細胞各自表現較高的 MMP-9 以及 MMP-1，此為胞 外基質成分重組所需的蛋白切割酵素，表明其對於移植細胞之微環境重建之特質 差異。以上結果進一步說明本實驗室過去動物實驗比對間質幹細胞輔助 CD34+
造血幹細胞移植成功率之差異結果。本實驗由基因體表現比較分析的結果顯示了 不同組織來源間質幹細胞之間的差異性與共通性，這些分子訊息可提供我們更進 一步探討並了解不同組織間質幹細胞特性，在未來幹細胞醫療以及組織修復的應 用上也是重要的參考依據。
關鍵字: 組織間質幹細胞，人類互補去氧核糖核酸微矩陣列
英文摘要
Human tissue mesenchymal stem cells (MSCs) have been initially identified in adult bone marrow and in other tissues. Although these cells shared similar morphology, cell surface markers, and the potential to differentiate into mesenchymal lineages, their proper specificity is poorly defined. In order to specify the proper characteristics of tissue mesenchymal stem cells, we isolated mesenchymal stem cells from adipose tissue (Adipose-derived mesenchymal stem cells ; AD-MSCs) to compare with that of bone marrow tissue (Bone marrow mesenchymal stem cells ; BM-MSCs) and
conducted the functional genomic analyses of these two stem cell populations by means of human cDNA microarray analysis and semi-quantitative RT-PCR. The analytical results indicated that BM-MSCs and AD-MSCs shared the common gene expression involved in early development and multipotent differentiation potential.
The distinctions between these two tissue MSCs can be identified in their cell-cell interactions and tissue supportive function. By RT-PCR we further confirmed that various genes of cytokines, growth factors, and ligands were significantly expressed differentially in these two MSC populations. BM-MSCs preferentially expressed LIF, SDF-1, BMP-4, PlGF-1, PlGF-2, Jagged-1, HGF, and HB-EGF gene expressions while AD-MSCs expressed more the MMP-1 gene only. The LIF, SDF-1, HGF
proteins expression were confirmed by Western blotting analyses. These results reflect the functional discrepancy in the properties of homing and maintaining the stem cells hematopoiesis and angiogenesis. This study further elucidate the results of our previous finding in a in vivo study of BM-MSCs supporting HSCs engraftment in NOD/SCID mice but AD-MSCs not. In this study we demonstrated that the tissue specificity of mesenchymal stem cells can be identified by the molecular
characterization of their gene and protein expression profiles. This approach has indispensable value for further insight into the nature of tissue stem cell populations in addition to their cell surface markers descriptions and multipotency examinations in vitro. Molecular information uncovered in this study may provide important cues for mesenchymal stem cell based applications.
Key words: tissue mesenchymal stem cells, human cDNA microarray
