臺灣博碩士論文加值系統

骨質疏鬆為骨骼質量降低及細微構造損傷的骨骼疾病，會造成骨骼容易碎裂，增加骨折發生的危險。隨著年齡的增加，骨髓內的脂肪細胞也隨之增加，並降低造骨細胞的形成，進而導致骨質疏鬆。骨質疏鬆症患者其骨髓內脂肪細胞的量與大小均增加，脂肪細胞過多而使造骨細胞不足，是可能造成骨質疏鬆的原因之一。已有研究證實，大豆異黃酮素﹙soy isoflavones﹚植物性雌激素可以預防骨質疏鬆症、動脈粥狀硬化及肥胖。咖啡因是一種植物性黃嘌呤生物鹼化合物，可以抑制由胰島素刺激之大鼠脂肪細胞造成的肥胖。脂肪基質幹細胞具有分化成不同細胞型態的能力。然而，大豆異黃酮素及咖啡因如何調控基質幹細胞的分化目前仍未被完全了解。因此本研究是利用脂肪基質幹細胞具分化之特性，來探討大豆異黃酮素及咖啡因對基質幹細胞分化為造骨細胞及脂肪細胞之影響。
首先，將分離出來的脂肪基質幹細胞，分別加入促脂肪及造骨細胞分化液，確認取得之細胞具有分化成脂肪及造骨細胞的能力。接著，在基質幹細胞進行分化期間，加入不同濃度的大豆異黃酮素及咖啡因，結果顯示，在低濃度大豆異黃酮素（0.3 μM）及咖啡因 （0.1 mM）作用下，不但可以降低細胞質內三酸甘油脂的累積和基質幹細胞分化為脂肪細胞，亦可增加調控造骨細胞分化之相關蛋白質Runx2、Sirt1及RhoA之表現量及細胞礦物質堆積；然而，當大豆異黃酮素（10 μM）在高濃度時，造成細胞質內三酸甘油脂的堆積增加，同時脂肪細胞分化調控因子PPAR-γ2，脂肪細胞激素leptin mRNA表現量也增加，並減少Runx2、Sirt1及RhoA之表現量及細胞礦物質堆積之情形。而在咖啡因（1 mM）高濃度作用下，雖然可以降低細胞質內三酸甘油脂的累積，但同時也降低了Runx2、Sirt1及RhoA之表現量及細胞礦物質堆積之情形。此外，大豆異黃酮素濃度在0.3 μM、咖啡因在0.1 mM時，不但增加蝕骨細胞抑制因子OPG的表現外，也能有效抑制骨再吸收因子TNF-α、IL-6 及OPN的釋放。這些結果說明大豆異黃酮素及咖啡因在低濃度作用下，可以增加基質幹細胞分化形成造骨細胞而降低脂肪細胞的分化，但在高濃度作用則相反，在大豆異黃酮素高濃度作用下，會增加基質幹細胞分化形成脂肪細胞。因此，每日適量的攝取大豆異黃酮素及咖啡因有助於防止基質幹細胞分化形成脂肪細胞，藉此達到防止骨質疏鬆症之效果。

Osteoporosis is defined as a skeletal disorder characterized by low bone mass andmicroarchitectural deterioration of bone tissue, leading to enhanced bone fragility and aconsequent increase in fracture risk. Aging and osteoporosis cause a decrease in the number of bone-forming osteoblasts and an increase in the number of marrow adipocytes. In addition, early histomorphometric observations suggested that the number and size of marrow adipocytes increase in a linear manner with age. All these evidences clearly demonstrate the reciprocal relationship between osteoblast and adipocyte differentiation. The phytochemicals such as soy isoflavones play an important role in prevention of many diseases including osteoporosis, atherosclerotic and obesity. Caffeine, a plant xanthine alkaloid compound, is one of the major components in tea, coffee, and carbonated drinks. In the present study, caffeine inhibits insulin-induced glucose uptake in rat adipocytes and affects bone metabolism. By cloning studies, they have shown that some adipose-derived stem cells (ADSCs) have multilineage differentiation potential. However, the molecular mechanism of soy isoflavones and caffeine on mesenchymal stem cells differentiation has not been full elucidated. Therefore, the objective of this study investigate the effects of soy isoflavones and caffeine on adipogenic and osteogenic differentiations of stromal stem cells.
In this study, our resulted indicates that ADSCs have been successfully isolated and induced differentiation into adipocytes and osteoblasts. During stromal stem cells differentiate into adipocytes and osteoblasts treated with lower soy isoflavones dose (0.3 μM) and caffeine (0.1 mM), there were decreased on lipid droplet accumulation through Oil Red O and Nile red staining. And, there were increased the expressions of osteogenic transcriptional factor, Runx2, Sirt1 and Rho A. In addition, deposition of calcium was enhanced slightly by Alzarin Red S and Von Kossa staining. However, at higher soy isoflavones dose (10 μM), there were increased on adipogenic transcriptional factor, PPAR-γ2 and adipocytokine, leptin and cellular lipid droplt accumulation significantly. But, deposition of calcium and the expression of Runx2, Sirt1 and Rho A during osteoblast differentiation were inhibited. Morever, the PPARγ2, leptin mRNA expressions and lipid droplet accumulation during adipocyte differentiation were inhibited by treatment with caffeine. But there were decreased the expressions of Runx2, Sirt1 and Rho A and deposition of calcium. In addition, at 0.3 μM soy isoflavones dose and 0.1mM caffeine dose, there was not only increased the expression of osteoclastogenesis inhibitory factor, OPG but also inhibited the mRNA expressions of bone-resorbing factor, TNF-??, IL-6 and OPN. Taken together, these results suggested lower soy isoflavones and caffeine dose act on stromal stem cells to enhance osteoblast differentiation and inhibit adipocyte differentiation. In addition, they probably inhibited osteoclast activation through enhancing OPG and decreasing bone-resorbing factor. Conversely, higher soy isoflavones and caffeine dose act on stromal stem cells to inhibit osteoblast differentiation. Therefore, appropriate levels of daily soy isoflavones and caffeine intake via nutriention or additional supplementation might be helpful for prevention of increased stromal stem cells adipogenesis as seen in postmenopausal osteoporosis.

壹、中文摘要．．．．．．．．．．．．．．．．．．．9
貳、英文摘要．．．．．．．．．．．．．．．．．．．11
?礡B緒論．．．．．．．．．．．．．．．．．．．．．13
一、骨骼系統．．．．．．．．．．．．．．．．．．16
1. 骨骼簡介．．．．．．．．．．．．．．．．．16
2. 骨骼的結構．．．．．．．．．．．．．．．．16
3. 骨質的建立．．．．．．．．．．．．．．．．16
4. 骨骼再塑．．．．．．．．．．．．．．．．．17
5. 骨細胞的分類．．．．．．．．．．．．．．．19
(1) 骨細胞．．．．．．．．．．．．．．．．20
(2) 造骨細胞．．．．．．．．．．．．．．．20
I. 造骨細胞生長及分化過程中相關分子表．．．22
II. 骨礦化形成之機制．．．．．．．．．．．25
III. 造骨細胞誘導分化之培養液 ．．．．．．．26
(3) 蝕骨細胞 ．．．．．．．．．．．．．．．29
I. 調控蝕骨細胞生成及活化之相關分子．．．．．30
二、骨質疏鬆症．．．．．．．．．．．．．．．．33
1. 骨質疏鬆的定義．．．．．．．．．．．．．．34
2. 骨質疏鬆的分類．．．．．．．．．．．．．．35
3. 骨質疏鬆的原因．．．．．．．．．．．．．．36
4. 骨質疏鬆的治療．．．．．．．．．．．．．．37
三、肥胖和骨質疏鬆症的關係．．．．．．．．．．．．39
1. 骨髓中脂肪細胞和骨質疏鬆的關係．．．．．．．40
2. 造骨細胞和脂肪細胞生成的關係．．．．．．．．41
3. 調控造骨和脂肪細胞分化之轉錄因子．．．．．．42
四、脂肪細胞．．．．．．．．．．．．．．．．．．44
1. 脂肪細胞的分化．．．．．．．．．．．．．．44
2. 調控脂肪細胞分化之轉錄因子．．．．．．．．．46
3. 脂肪細胞激素．．．．．．．．．．．．．．．49
4. 脂肪細胞誘導分化之培養液．．．．．．．．．．52
五、幹細胞．．．．．．．．．．．．．．．．．．．54
1. 幹細胞的定義．．．．．．．．．．．．．．．54
2. 幹細胞的分類．．．．．．．．．．．．．．．54
3. 成體幹細胞．．．．．．．．．．．．．．．．56
4. 間葉幹細胞．．．．．．．．．．．．．．．．56
5. 脂肪幹細胞．．．．．．．．．．．．．．．．57
六、大豆異黃酮．．．．．．．．．．．．．．．．．59
1. Isoflavones的介紹及化學構造．．．．．．．．．59
2. Isoflavones的特性．．．．．．．．．．．．．．60
3. Isoflavones於生理保健之應用．．．．．．．．．61
七、咖啡因．．．．．．．．．．．．．．．．．．．63
1. 咖啡因的特性．．．．．．．．．．．．．．．63
2. 咖啡因之動力學．．．．．．．．．．．．．．64
3. 咖啡因的作用機轉．．．．．．．．．．．．．64
4. 咖啡因的優點．．．．．．．．．．．．．．．64
肆、研究目的．．．．．．．．．．．．．．．．．．．67
伍、研究設計．．．．．．．．．．．．．．．．．．．68
陸、實驗材料．．．．．．．．．．．．．．．．．．．69
一、 動物．．．．．．．．．．．．．．．．．．．69
二、 細胞．．．．．．．．．．．．．．．．．．．69
三、 材料．．．．．．．．．．．．．．．．．．．69
四、 藥品．．．．．．．．．．．．．．．．．．．73
五、 儀器與設備．．．．．．．．．．．．．．．．81
柒、實驗方法．．．．．．．．．．．．．．．．．．．84
一、 細胞培養．．．．．．．．．．．．．．．．．84
1. 解凍細胞．．．．．．．．．．．．．．．．．84
2. 繼代培養．．．．．．．．．．．．．．．．．84
3. 細胞計數．．．．．．．．．．．．．．．．．84
4. 冷凍細胞．．．．．．．．．．．．．．．．．85
二、 脂肪基質幹細胞分離．．．．．．．．．．．．．85
1. 脂肪組織的萃取．．．．．．．．．．．．．．86
2. 脂肪基質幹細胞的萃取及培養．．．．．．．．．87
三、 脂肪基質幹細胞的誘導分化．．．．．．．．．．87
1. 脂肪細胞的誘導分化．．．．．．．．．．．．．87
2. 造骨細胞的誘導分化．．．．．．．．．．．．．88
四、 M210B4細胞的誘導分化．．．．．．．．．．．．89
1. 脂肪細胞的誘導分化．．．．．．．．．．．．．89
2. 造骨細胞的誘導分化．．．．．．．．．．．．．90
五、 脂肪細胞之檢測．．．．．．．．．．．．．．．91
1. 油紅染色法．．．．．．．．．．．．．．．．91
2. 尼羅河紅染色法．．．．．．．．．．．．．．91
六、 骨礦化之檢測．．．．．．．．．．．．．．．92
1. 茜素紅染色法．．．．．．．．．．．．．．．92
2. Von Kossa stain．．．．．．．．．．．．．．．92
3. 鹼性磷酸?′V色法．．．．．．．．．．．．．93
4. 鹼性磷酸?′〝妠?定．．．．．．．．．．．．．94
七、 反轉錄聚合?○s鎖反應．．．．．．．．．．．．94
八、 西方墨點法．．．．．．．．．．．．．．．．97
捌、實驗結果．．．．．．．．．．．．．．．．．．．101
一、 脂肪基質幹細胞之取得及分化特性之探討．．．．．101
1. 脂肪基質幹細胞之特性及型態表現．．．．．．．101
2. 脂肪基質幹細胞誘導分化成脂肪細胞及造骨細胞之測
定．．．．．．．．．．．．．．．．．．．．102
二、 M210B4細胞誘導分化成脂肪細胞及造骨細胞及鑑定．．．．．．．．．．．．．．．．．．．．103
三、 大豆異黃酮素及咖啡因對於基質幹細胞誘導分化成脂肪細胞的影響．．．．．．．．．．．．．．．．104
四、 大豆異黃酮素及咖啡因對於基質幹細胞誘導分化成脂肪
五、 細胞之轉錄因子PPAR-γ2的影響．．．．．．．．106
六、 大豆異黃酮素及咖啡因對於脂肪細胞之細胞激素leptin的影響．．．．．．．．．．．．．．．．．．．108
七、 大豆異黃酮素及咖啡因對於基質幹細胞誘導分化成造骨細胞的影響．．．．．．．．．．．．．．．．108
八、 大豆異黃酮素及咖啡因對於脂肪基質幹細胞誘導分化成造骨細胞之轉錄因子的影響．．．．．．．．．．110
九、 大豆異黃酮素及咖啡因對於基質幹細胞誘導分化成造骨細胞之鹼性磷酸?〞獐v響．．．．．．．．．．．112
十、 大豆異黃酮素及咖啡因對於脂肪基質幹細胞誘導分化成造骨細胞之蝕骨細胞抑制因子OPG的影響．．．．．113
十一、 大豆異黃酮素及咖啡因對於脂肪基質幹細胞誘導分化成脂肪細胞之骨再吸收因子的影響．．．．．．．114
玖、討論．．．．．．．．．．．．．．．．．．．．．115
拾、參考文獻．．．．．．．．．．．．．．．．．．．120拾壹、圖表．．．．．．．．．．．．．．．．．．．．128
拾貳、附錄．．．．．．．．．．．．．．．．．．．． 163

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