- 學位論文
探討異黃酮素對造骨細胞與蝕骨細胞之生長、分化及生理活性之影響
The study of the effects of the isoflavones on the growth, differentiation and functional activity of osteoblasts and osteoclasts
摘要
骨質疏鬆症（osteoporosis）好發於女性，目前已知其肇因主要與老化及婦女於停經後之生理性缺乏雌激素有關。於相關研究報告中發現，雌激素對停經後婦女而言，是預防骨質疏鬆症最有效的藥物；但此一荷爾蒙替代療法(hormone replacement therapy，HRT)會增加子宮內膜癌和乳癌發生的機會。在臨床上已有許多證據顯示，植物性雌激素如異黃酮素（isoflavones），可增加女性骨質量，減緩骨質流失。目前，異黃酮素對於與骨質代謝相關之蝕骨細胞（osteoclast）與造骨細胞（osteoblast）生長之作用、鈣磷平衡之調節、骨質生成與骨質的再吸收作用之分子標誌及骨質密度消長之影響與其機轉，並不十分清楚。故本研究擬以異黃酮素對於骨質疏鬆症之預防及治療為目標。利用由Sprague- Dawley大鼠之幼鼠長骨及胎鼠顱頂骨分骨分離取得之蝕骨前趨細胞與造骨前趨細胞，並進一步分別以1,25(OH)2D3、dexamethasone與ascorbic acid之促分化培養模式來獲得大量成熟的蝕骨細胞與造骨細胞，試驗中所使用之異黃酮素（genistein或daidzein、biochanin A 或三種異黃酮素併用）濃度乃採用一般生理可達之異黃酮素濃度（10-8、10-7與10-6 M）；依實驗設計，首先分別測定藥物處理後對蝕骨細胞之前趨細胞（pre-osteoclast）與造骨細胞之前趨細胞（pre-osteoblast）生長的影響，並利用蝕骨細胞之分化及成熟期所表現的酸性磷酸酶（acid phosphatase；AP）及造骨細胞分化及成熟期所表現的鹼性磷酸鹽酶（alkaline phosphatase；ALP）來測定異黃酮素對蝕骨細胞之前趨細胞與造骨細胞之前趨細胞分化成熟為蝕骨細胞及造骨細胞之影響；更進一步的，測定異黃酮素對於蝕骨細胞表現與骨質再吸收作用相關之酸性磷酸酶活性及對造骨細胞表現骨質生成作用相關之鹼性磷酸鹽酶活性的影響，並進一步分析異黃酮素對造骨細胞礦化作用（calcification）之形成效益與造骨細胞之前趨細胞之細胞移行作用（migration）的影響。由目前研究結果顯示，三種異黃酮素於10-6 M之藥物濃度下，均可促進造骨細胞之前趨細胞之增生並分化為成熟的造骨細胞，並促進成熟造骨細胞之骨生成作用；而三種異黃酮素於10-6 M之藥物濃度下，均可抑制蝕骨細胞之前趨細胞的生長與分化成熟，以及抑制成熟蝕骨細胞之骨再吸收作用；另外，於礦化作用形成效益之影響分析試驗中之結果顯示，三種異黃酮素於10-6 M之藥物濃度下，均可促進造骨細胞礦化結節的形成，其中以biochanin A或合併三種異黃酮素處理之實驗組的促進效果尤其顯著。而於異黃酮素對造骨細胞之前趨細胞之細胞移行影響實驗結果可知，三種異黃酮素於10-6 M之藥物濃度下，只有三種異黃酮素並用作用下才有明顯促進造骨細胞之前趨細胞之細胞移行的趨性。由我們的研究成果證實，異黃酮素能促進造骨細胞增生及成熟並抑制蝕骨細胞之生成來達到調節骨質代謝之作用。
並列摘要
Menopausal hormone decline contributes significantly to increase the risk of osteoporosis. Osteoporosis, typically reflects an imbalance in skeletal turnover lead to bone resorption exceeds bone formation. Bone resorption and bone formation respectively that is the unique function of the osteoclast and osteoblast, and anti-osteoporosis therapy to date has targeted these cells in osteoporosis. In clinical treatments, although estrogen replacement therapy (HRT) has been a widely used approach to prevent or treat post-menopausal osteoporosis, HRT increase the risk of breast and uterine cancer and many have other undesirable side effects. Recently, the natural phytoestrogens such as soy or red clover isoflavones exert estrogen-like activities and play a potential role in osteoporosis prevention and treatment. Dietary isoflavones have been shown to improve bone mass in human and rodent studies. However, the mechanism of action of isoflavones has not been fully clarified. Thus, the present study was undertaken to investigate the effects of isoflavones on primary osteoclasts and osteoblasts proliferation, differentiation and formation and clarify cellular mechanisms of isoflavones enhacement of bone formation and inhibition of bone resorption. Study design: Primary pre-osteoblasts were isolated from fetal rat calvaria and induced by ascorbic acid and primary pre-osteoclasts were isolated from neonatal rat long bones and induced by 1,25(OH)2D3. We evaluated the effects of isoflavones at various concentrations on pre-osteoblasts, osteoblasts, pre-osteoclasts and osteoclasts proliferation by assessing the crystal violet assay. In the differentiation assay, pre-osteoblasts, osteoblasts, pre-osteoclasts and osteoclasts were exposed to isoflavones（genistein or daidzein or biochanin A or mixture above） at various concentrations（10-8 to 10-6 M ）, then ALP-positive cells will be counted as osteoblasts and tartrate-resistant acid phosphatase （TRAP）-positive cells with more nuclei will be counted as osteoclasts were determinated, level of alkaline phosphatase (ALP) secreted by osteoblasts, acid phosphatase (AP) secreted by osteoclasts, phosphorus calcium deposition of osteoblasts and cell mobility of pre-osteoblasts were evaluated. Results: The results showed that isoflavones induced pre-osteoblasts proliferation and inhibited pre-osteoclasts and osteoclasts proliferation in dose-dependent manner. In osteoblasts, the number of ALP-positive cells, level of ALP, phosphorus calcium deposition and cell mobility increased depending on the physiological concentration of isoflavones. In osteoclasts, the number of TRAP-positive cells, level of AP decreased depending on the physiological concentration of isoflavones. Conclusions: These results suggest that the induction effects of isoflavones on osteoblasts proliferation and differentiation and suppressive effect on osteoclasts proliferation and differentiation might be one of the most important factors contributing to induction effect on bone repair and remodeling in vivo. Our results to suggest isoflavones can prevent bone resorption diseases by the promotion of bone formation and the prevention of bone resorption.