| 摘要: | 中藥對骨細胞活性的評估 蔡宏銘 指導教授 ： 陳悅生博士 中國醫藥學院 中國醫學研究所 由於傳統的中醫藥對於骨傷有很好的促進修復作用，所以在這實驗中我們利用骨細胞的培養，來探討龜鹿膠、杜仲、骨碎補、續斷、桑寄生、懷牛膝、菟絲子7種藥物對骨細胞的生化機轉。 將取自Sprague-Dawley 小鼠的頭蓋骨和脾臟分別做骨母細胞、蝕骨細胞和骨母細胞 /蝕骨細胞共培養，分別加入7種不同濃度的中藥粹取液，先用Liu’s染色和鹼性磷酸酵素( Alkaline Phosphatase, ALP )染色觀察其細胞型態以評估藥物的生物適應性，再利用MTT分析來評估中藥對骨細胞的存活和增生能力，以及分析其ALP活性、礦物質化結節( Mineralized Nodule )的形成和酒石酸耐受性酸性磷酸酵素( Ttartrate-Resistant Acid Phosphatase, TRAP ) 活性等生化指標，來評估骨母細胞和蝕骨細胞的分化能力。另外，利用TRAP染色進一步評估蝕骨細胞的分化型態。 實驗結果顯示，由Liu’s染色和 ALP染色細胞型態評估，除了濃度100 mg/mL外，其餘7種藥物6種濃度對骨細胞培養生物適應性均良好。另外評估礦物質化結節的形成，發現和骨細胞的分化能力有正相關。最後觀察龜鹿膠和菟絲子對單一骨母細胞培養和骨母細胞 / 蝕骨細胞共培養的MTT 分析和ALP活性均為促進效果，對蝕骨細胞TRAP活性為抑制效果。而桑寄生和懷牛膝則剛好相反。另外，杜仲、骨碎補和續斷對單一骨母細胞培養和骨母細胞 / 蝕骨細胞共培養的MTT 分析 和ALP活性在高濃度10 mg/mL和1 mg/mL下為抑制效果，而其他濃度則為促進效果，對蝕骨細胞TRAP活性在高濃度10 mg/mL和1 mg/mL下則為促進效果。 關鍵字：骨母細胞，蝕骨細胞，鹼性磷酸酵素，酒石酸耐受性酸性磷酸酵素， 礦物質化結節，細胞培養。; Evaluation of Chinese Herbal Medicines on Bone Cell Activities Hung-Ming Tsai Major Professor：Yueh-Sheng Chen Institute of Chinese Medical Science, China Medical College The traditional Chinese medicine has been reported as a good enhancer for bone healing. In this experiment, we investigated the biochemical effects of Gui-Lu-Jiao, Du-Zhong, Gu-Sui-Bu, Xu-Duan, San-Gji-Sheng, Hui-Niu-Xi and Tu-Si-Zi on cultured bone cells. In this study, we used Liu’s stain, alkaline phosphatase stain (ALP stain)and tartrate-resistant acid phosphatase stain (TRAP stain)to observe the morphological change in osteoblasts, osteoclasts and osteoblast/osteoclast co-cultures after they were mixed with different concentrations of the herbal medicine. In addition, we used the MTT assay to observe the survival and proliferation activity of bone cells. The differentiation effect of osteoblasts was evaluated with ALP activity and formation of mineralized nodules. Finally, the effect of herbal medicine on the differentiation of osteoclasts was evaluated with TRAP activity. Through the observations of the morphologies of nuclear and cytoplasm stained with Liu’s stain and ALP stain, we found that all the concentrations but the 100 mg/mL of the seven herbal medicine had no cytotoxicity. In addition, it was noted that the differentiation of osteoblasts was positively correlated with the formation of mineralized nodules. We also found that both the Gui-Lu-Jiao and Tu-Si-Zi wered promote the proliferation and differentiation of the osteoblasts and the osteoblast/osteoclast co-culture through the MTT assay and ALP activity analysis. However, both the herbal medicine could inhibit the TRAP activity of the osteoclasts. Reversed results were seen on the medicine was changed to San-Gji-Sheng and Hui-Niu-Xi. As for the Du-Zhong Gu-Sui-Bu and Xu-Duan, when their concentrations were highen than 1 mg/mL, the proliferation and differentiation of the osteoblasts and the osteoblast/osteoclast co-culture were inhibited. But, the high concentration of these medicine could accelerate the TRAP activity of the osteoclasts. Key word：Osteoblast (O.B.)；Osteoclast (O.C.)；Alkaline Phosphatase (ALP)； Tartrate-Resistant acid Phosphatase (TRAP)；Mineralized Nodule； Cell Culture |
