| 摘要: | 在骨骼生合成的平衡機制中，骨母細胞(Osteoblasts)及蝕骨細胞(Osteoclasts)扮演互相影響同時互相拮抗的角色。引發骨質疏鬆症的原因中，骨母細胞及蝕骨細胞的不平衡往往就是致病的關鍵:骨母細胞的不足或蝕骨細胞的過度活化。雌激素療法是現在廣泛用於治療骨質疏鬆症的治療方式，但是許多實驗報告都顯示雌激素療法有引發癌症的可能性。植物雌激素(phytoestrogen)是存在植物中的物質，對於雌激素接受器(estrogen receptor)有促進或拮抗的作用，以植物雌激素來取代雌激素治療骨質疏鬆症的可能性極待評估中。
在本研究中，選用植物雌激素三大分類中的Coumestrol藥物，研究在投予coumestrol的影響下，對由骨母細胞促進單核球細胞融合成為多核蝕骨細胞的機制受到的改變。研究選用八個月以上的ICR成鼠培養蝕骨細胞，以得自其四肢骨的骨母細胞和得自骨髓的單核球細胞來培養蝕骨細胞，希望能建立類似老年停經後婦女的體外培養生理模式。
由培養過程得知，因為使用的是相當於人類老年的母鼠，所以骨母細胞的活性極差，要利用骨母細胞刺激單核球培養蝕骨細胞，培養的時間點要相對的拉長，和一般培養蝕骨細胞過程約10天完成相比，必須有給予骨母細胞生長的時間，因此過程須延長到約14天。實驗選用Coumestrol濃度10-5M、10-6M、10-7M、10-8M、10-9M、10-10M，在細胞活性分析MTT test中，顯示在骨母細胞及蝕骨細胞共同培養模式中，投予各濃度的Coumestrol與控制組都有相同的細胞活性，所以可以知道投予Coumestrol對於細胞沒有細胞傷害產生，在這些藥物濃度範圍之下對於骨細胞都是安全的。利用針對蝕骨細胞的抗酒石酸性磷酸酶測試(ACP test)則發現，蝕骨細胞在10-5M、10-6M、10-7M濃度下ACP的活性有高於控制組的趨勢，這表示投與Coumestrol會提高蝕骨細胞的活性或是增加蝕骨細胞的數量。利用抗酒石酸性磷酸酶染色來觀察，蝕骨細胞為多核巨大的細胞，以刺激發炎反應的LPS來投與在培養的蝕骨細胞中時，在第6天投與LPS會使蝕骨細胞有變大變多的現象發生，如果是在第三天就投與LPS反而會使蝕骨細胞的形成受到抑制。
以Coumestrol10-7M為投予濃度，做骨頭細胞mRNA等級real time RT-PCR，顯示此共同培養系統之中，LPS會增加IL-1、IL-6，以及使促進蝕骨細胞生長的RANKL表現加強，並且降低抑制蝕骨細胞成熟的OPG的表現，因此會使蝕骨細胞的生長增加。而coumestrol不論在LPS有否存在的情況下，也都會使IL-1、IL-6，以及RANKL的表現加強，比較不同的是coumestrol也同時會使抑制蝕骨細胞生長的護骨素OPG表現加強。而coumestrol對於雌激素受體ER-α以及ER-β的表現都有加強，可是ER-α的表現增強約為兩倍， ER-β的表現增強約為3-4倍，顯示了coumestrol較傾向於作用於ER-β。由這些實驗結果顯示，coumestrol有使骨母細胞加強的能力，但是也同時有讓有利於蝕骨細胞生長的IL-1、IL-6，RANKL增加的作用，顯示了coumestrol有使蝕骨細胞生長加強的可能性。因此對於使用coumestrol對於骨質疏鬆的治療，或許還須評估其可行性。
In bone remodeling, the balance between bone formation and resorption is determined by numbers and activity of osteoblast and osteoclast. Osteoporosis results from decrease in osteoblast activity and relative increase of osteoclast activity. Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decreases after menopause and the estrogen deficiency results in bone loss. There are, however, risks associated with estrogen therapy. Phytoestrogens, which have in part some structural similarity to 17beta-estradiol, are reported to act as agonists/antagonists of estrogen in animals and humans. Phytoestrogen therapy for menopausal osteoporosis is need to be evaluated.
In this study, we want to assess the effects of coumestrol , a kind of phytoestrogens ,on osteoblast/monocyte coculture model system to the formation of osteoclasts. Both the osteoblast and monocyte come from 8 month female neonatal mice ,in order to establish a model of bone system similar to menopausal women.
In this coculture system, the formation of mature osteoclast is about 14 days. To investigate the effect of coumestrol on osteoblasts and osteoclasts, the bone cell activity was examined by MTT assay , and showed no difference with coumestrol (10-5M~10-12M). It suggests that coumestrol will not cause cell damage. The enzyme activities of osteoblasts and osteoclasts were measured by the analysis of ALP and ACP. The results showed that coumestrol 10-5M~10-7M both increase the osteobasts and osteoclasts activities.
Futher more, we had tested this coculture system with coumestrol 10-7M and lipopolysaccharide, then assessed the cellular expression of osteoclast formation related gene IL-1,IL-6,RANKL ,OPG, and estrogen receptors (ER-αand ER-β) by real-time PCR. IL-1, IL-6 and RANKL are the genes that enhance osteoclast formation and OPG is the gene to limit osteoclast formation. Lipopolysaccharide stimulated the mRNA expression of IL-1, IL-6, RANKL, and inhibited the expression of OPG, showed the ability to increase osteoclast formation. Coumestrol also stimulated the mRNA expression of IL-1, IL-6, RANKL, showed the possibility to increase osteoclast formation , but coumestrol could stimulate OPG expression at the same time. In the test of estrogen receptor (ER-αand ER-β), coumestrol both increased the expression of ER-αand ER-β. Coumestrol increased mRNA expression of ER-α by two times, and increased mRNA expression of ER-β by three or four times, showed that coumestrol has higher affinity to ER-βand weaker to ER-α.
These results present that coumestrol both increase osteoblast and osteoclast activities, so the use of coumestrol to treat osteoporosis may still need further evaluation. |
