探討大豆異黃酮素及維生素D對去除卵巢 大鼠之骨質密度與生理活性之影響

The study of the efficacy of soy-isoflavones and vitamin D on bone mineral density and biological activities in ovariectomized rats

許恆瑋(Hen-Wei Hsu)

指導教授 ： 張基隆

高雄醫學大學/醫學院/生物化學研究所/碩士(2005年)

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，植物性雌激素如大豆異黃酮素（isoflavones），可增加女性骨質量，減緩骨質流失。本研究以大鼠切除卵巢的方式模擬女性停經後之生理狀況，探討大豆異黃酮素對去除卵巢大鼠之骨質密度與生理活性之影響。實驗設計方面，我們先將大鼠卵巢切除後，再分別投予17b- estradiol、soy extracts（含 isoflavones）或soy extracts 添加維生素D，每組各6隻大鼠，共餵食14週。實驗測定方面，我們利用雙能量X光測定儀（DEXA）測定大鼠骨質密度的變化；並利用 ELISA kit 測定血清中鹼性磷酸酶之活性及骨鈣素、tumor necrosis factor-a（TNF-a） 和 interleukin-1b（IL-1b）的含量；而後，以 RT-PCR 技術探討大鼠骨組織造骨細胞（osteoblast）相關分子標誌之基因表現變化及蝕骨細胞（osteoclast）相關分子標誌之基因表現變化。最後，再作大鼠股骨組織切片染色（H/E stain）分析，觀察骨質生理之變化。實驗進行14週後，結果顯示出共同投予大豆異黃酮素或併用維生素D，在骨密度的改善上有近似於投予雌激素的效果，且和 OVX 組有顯著差異；在大鼠血清測定方面，共同投予大豆異黃酮素或併用維生素D，能促進鹼性磷酸酶之活性，提高骨鈣素的含量；相對的，能降低 TNF-a 和 IL-1b 的含量；在骨組織萃取 mRNA 方面，投予大豆異黃酮素或併用維生素D後，可活化造骨細胞相關基因Osterix、collagen type I、alkaline phosphatase（ALP）、osteocalcin、osteoprotegerin（OPG）及抑制蝕骨細胞相關基因receptor activator of NF-kB（RANKL）和tartrate-resistant acid phosphatase（TRAP）的表現；在股骨組織切片染色方面，可觀察到投予大豆異黃酮素或併用維生素D後，其股骨頭骨小樑（trabecular bone）的流失獲得明顯的改善。依現階段的研究成果初步證實，異黃酮素確有調節骨質代謝的效果，可能是藉由抑制蝕骨細胞的活性來降低骨質流失，且可活化造骨細胞，促進骨質生成；與維生素D併用可能有更好的效果。

關鍵字

卵巢切除術 ； 異黃酮素 ； 骨質疏鬆症 ； 造骨細胞 ； 蝕骨細胞 ； 鹼性磷酸?

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, natural alternatives such as soy isoflavones exert estrogen-like activities and play a potential role in osteoporosis to help prevent postmenopausal osteoporosis. Genistein and daidzein are the most abundant isoflavones present in soy. In this study, we use ovariectomized (OVX) rats as experimental model of estrogen depletion- induced bone loss; we examine the therapeutic efficacy of soy isoflavones and combination of vitamin D（VitD）on changes of femur bone mineral density (BMD) and biological activity in OVX Sprague-Dawley rats. The rats were randomly assigned to several groups: (1) sham-operated (2) OVX（ovariectomy）(3) OVX treated with 17b-estradiol (4) OVX treated with soy extracts（containing isoflavones）(5) OVX treated with soy extracts and 67 I.U. Vit D. Each group had six rats and treated for 14 weeks. Bone mineral density and trabecular bone volume of distal femurs were measured to verify the osteopenia of bones. Rat serum alkaline phosphatase and osteocalcin were measure to detect osteoblasts activity. Rat serum TNF-a
and IL-1b could promote osteoclast activation, so they were measured to detect osteoclast activation. The mRNA expressions of osterix, type I collagen, alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin（OPG）in bones were measured as markers of osteoblasts；receptor-activator of NF-kB ligand（RANKL）and tartrate-resistant acid phosphatase（TRAP）in bones were measured as markers of osteoclasts. The results showed that isoflavones can reduce bone loss according to BMD analysis and bone histomophometry. On the other hand, isoflavones can increase mRNA expression of osterix、type I collagen、ALP、osteocalcin、OPG in osteoblasts, decrease mRNA expression of RANKL and TRAP in osteoclasts. Our results show that isoflavones can prevent bone resorption diseases by the promotion of osteoblast activation（bone formation）and the prevention of osteoclast activation（bone resorption）. Combining isoflavones with vitamin D even have better effects.

並列關鍵字

alkaline phosphatase ； ovariectomy ； osteoporosis ； isoflavone ； osteoblast ； osteoclast

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