年老骨髓間質幹細胞功能性基因體變化之研究

年老骨髓間質幹細胞功能性基因體變化之研究

A Study on Functional Genomic Changes of Bone Marrow Mesenchymal Stem Cells in Elder People

 之前的臨床生理研究發現了人類隨著老化會導致骨質流失的現象，但是目前對於其細胞生理以及分子調控詳細機制 並不清楚。

骨髓中含有一種骨髓間質幹細胞，它是成骨細胞以及成脂細胞的共同前驅細胞。目前已知在老年人的骨髓檢體中，

可以發現到較多油脂堆積，相對的骨質形成功能也有減少，我們認為成脂細胞和成骨細胞可能會有平衡競爭的現象

，但目前對於骨髓間質幹細胞分化各個路徑詳細的調控機制及分子傳遞路徑並不十分清楚。

骨質重建 (Bone remodeling) 簡單來說包涵骨骼之溶蝕作用 (bone resorption), 及隨之而來之骨骼生成作用 (bone form ation) 。骨質重建之所以能順利進行，必須要骨骼中二種不同之族群細胞通力合作才行。這二種細胞為負責骨骼溶蝕 作用之蝕骨細胞 (osteoclast) 及負責骨形成作用之成骨細胞 (osteoblast) 。這二種細胞間之協調如果出現失衡現象，將 導致骨重建之失衡，而出現臨床上常見之骨質流失。成骨細胞又是從骨髓間質幹細胞分化而來 ; 而蝕骨細胞蝕從造 血幹細胞分化而來，所以我們為了了解在同樣的骨髓維環境中的這兩種細胞，是否會透過分泌某些激素來達到互相 調控的效果，使得骨質重建的機制受到影響。

所以本實驗藉由取自不同年齡的骨髓間質幹細胞檢體，分析其成骨及脂肪分化相關轉錄因子基因的變化，我們將骨 髓細胞檢體分為兩大族群，將 20 到 40 歲的檢體設定成年輕族群 ;60 到 80 歲的檢體設定成年老族群，再進一步去針 對其分化功能做比較。我們可以觀察到從年老檢體分離出來的骨髓間質幹細胞，明顯有往脂肪油滴分化的傾向 ; 而 從年輕檢體所分離出來的骨髓間質幹細胞，則是有較高的鈣質沉積的現象。

接著利用半定量反轉錄聚合 ? 連鎖反應去觀察一些調控分化及早期基因的表現情形，其中我們發現 Hepatocyte growt h factor (HGF) 會隨著年紀的增加有升高表現的情形，而 HGF 在於骨質形成的相關功能所扮演的角色，可以促進蝕 骨細胞的成熟及生長，而且會吸引蝕骨細胞往骨質溶蝕的部位吸引，這對於蝕骨的作用有相當大的影響性。

最後利用了人類細胞激素蛋白表達微陣列 (Human Cytokine Antibody Array) 去偵測從不同年齡檢體分離出骨髓間質 幹細胞的培養液，其分泌激素的改變情形，發現 IL-6 和 MCP-1 明顯在年老檢體中培養液較多，它們被指出可以促 進蝕骨前驅細胞的成熟。這種現象可能影響同在骨髓微環境中造血幹細胞 (HSC) 分化成各種血球系細胞調控之機制 受到影響，而促使得造血幹細胞往蝕骨細胞的分化能力有所變化，而骨骼重建 (bone remodeling) 的平衡失衡造成老 年的骨質流失增加的情形。

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Clinical and histomorphometric studies have shown that aging is associated with decreased bone mass and bone formation potential. However, the cellular and molecular mechanisms underlying the decreased bone formation potential are under explored.

In the marrow, stromal osteoblasts and adipocytes share a common precursor, bone marrow mesenchymal s tem cells. It is well known the volume of marrow adipocyte increases with a decreased bone forming poten tial in the elder marrow. This phenomenon may be resulted from a comparatively favor of adipogenesis an d diminished ability of osteoblastogenesis in the elder’s BM-MSCs. Therefore, it is reasonable to assume t he equilibrium of osteogenic and adipogeneic potential in marrow may be shifted by the aging of MSCs. H owever, the molecular basis of the equilibrium shift is unclear.

To examine the equilibrium in the aged BM-MSC, we compared the osteogenic and adipogenic differentiat ion potentials of BM-MSCs isolated from healthy young (age 20-40, n =4) and elder (age 60-80, n = 4) mar rows. The osteogenic potential is obviously higher in BM-MSC of young donors than the elder ones. This phenomenon was demonstrated by the Von Kossa’s Staining and calcium precipitate quantitation in the cul ture .

To identify the transcriptional changes of the osteoclast formation genes, semi-quantitative RT-PCR were performed, and increased expression of hepatocyte growth factor (HGF) mRNA were found in the elder do nors. Keeping in line with the upregulation of HGF gene in the elder BM-MSC, HGF have been known as a major factor controlling both migration and proliferation of osteoclasts at the sites of bone resorption.

Furthermore , by using Human Cytokine Antibody Array analysis, we found IL-6 and MCP-1 were increas

ed in the aged donors. These osteotropic factors that induce osteoclast formation act indirectly by binding

marrow stromal cells, which in turn induce upregulation of RANK ligand expression.