壯筋續骨湯對骨細胞活性之影響
壯筋續骨湯為一種常用於促進骨折癒合之中藥複方，當中包括 17 種中藥材。根據傳統之中醫理論
，這些組成物具有補腎強精、促進血液循環、幫助胃腸道吸收等功能。而在本實驗中擬藉由骨細胞 培養之模式，探討此一複方對骨形成作用和骨吸收作用之影響，並以各種生化分析之方法來印證其 作用機制。
中藥複方經水煮萃取，將之濃縮乾燥，針對此一初萃產物進行各項活性分析。並進一步以乙酸乙酯 (ethyl acetate) 分配成為有機層和水層。同樣在取得新生鼠之骨母細胞和蝕骨細胞後，分別加入有機 層和水層作為實驗組以判讀其活性。並且在不同之時間點，進行細胞存活率測試 (MTT assay) 、鹼 性磷酸酵素 (ALP) 、 酸性磷酸酵素 (ACP) 、 乳酸脫氫酵素 (LDH) 、 總蛋白質含量 (total pr otein content) 等酵素活性測試、礦化染色實驗 (von Kossa stain) 和抗酒石酸磷酸酵素染色試驗 (T RAP stain) 以觀測其對骨母細胞分化成熟及蝕骨細胞之生成過程的影響。另外以抽取骨母細胞中之 mRNA 之方式檢視骨母細胞之特異性蛋白質表現。
在細胞存活率方面之結果顯示，複方初萃物在濃度為 100 mg/mL 時確實能有效加強骨母細胞增生 期之活性表現，並對鹼性磷酸酵素之分泌能力無激發作用，顯示骨母細胞族系在此藥物作用下，多 數細胞維持在較早期之型態而不斷分裂，因而延遲其進入分化期之速度。在進行分配後之水層在濃 度為 1mg/mL 時促進骨母細胞增生之效果高達 46% ，並且對蝕骨細胞亦有 20% 以上之抑制效果
，呈現與原初萃物相同走勢。而再從鹼性磷酸酵素活性以及礦化染色結果對照，可以推斷水層藥物 於骨形成作用之早期階段有顯著之促進作用。最後再比對骨母細胞特異性蛋白質之 mRNA 色帶強 度，發現 Col-I 及 OPN 的表現皆有明顯之差異，因此更能夠證實此一論點。在酸性磷酸酵素活性方 面，實驗組於所有時間點之酵素活性皆與控制組達 50% 以上之差距，除了與蝕骨細胞存活率之結 果相呼應外， TRAP 染色定性實驗也觀察到水層藥物能明顯抑制蝕骨細胞之生成。
因此，壯筋續骨湯水層具有使早期骨母細胞分裂能力趨於活躍而加強骨形成作用；而對蝕骨細胞之 生成和活化皆有抑制作用，因而達到抑制骨吸收作用之效果。
Influence of Chuang-Chin-Hsu-Ku-Tang on Bone Cell Activities
The traditional Chinese medical prescription : Chuang -Chin-Hsu-Ku-Tang contains seventeen kinds of Chin ese herbal drugs and is usually used to accelerate fracture healing. According to the theory of traditional Chi nese medicine, these Chinese medicines may warm the kidney, promote blood circulation and improve gastr ointestinal absorption; however, action mechanisms for their effects have not been identified. The purpose of this study is to evaluate and elucidate the effect of Chuang-Chin-Hsu-Ku-Tang on osteoblasts and osteoclasts via in vitro cell cultures.
The herb medicine was extracted with boiling water and then partitioned with ethyl acetate. Primary culture cells were isolated from newborn Wistar rats. In the in-vitro study, the effects of water and EtOAc extrat on cell viability were determined by the method of MTT assay during the time course. The enzyme activities of osteoblasts and osteoclasts were measured by the analysis of alkaline phosphatase (ALP), acid phosphatase (ACP), lactate dehydrogenase (LDH) and total protein content. The differentiation of osteoblasts and osteocl asts was analyzed by von Kossa stain and tartrate-resistant acid phosphatase stain, respectively. Moreover, w e try to find the specific protein by extracting mRNA from osteoblast.
The cell survival test showed crude, extract at concentration 100 mg/mL, enhancing the activity of osteoblast during proliferation stage, but no effect on ALP secretion. Obviously, the effect of herb medicine on osteobl ast, keep the cell in proliferation as in early stage, but delay turning to differentiation. After partition, the wat er layer at concentration 1 mg/mL promoting osteoblast proliferation over 46%, but inhibiting osteoclast mor e than 20%, showed the same bioactivity as crude extract. From ALP and von Kossa stain test, the water laye r extract promoted the bone formation in the early stage. Comparing with osteoblast specific protein, mRNA band intensity, those differences of Collagen type I and Osteopontin were found. The enzyme activity of AC P showed more than 50% difference between the experimental and control, the same as TRAP stain showed.
