利用斑馬魚尾鰭再生探討雙磷酸鹽藥物造成顎骨壞死的致病機制-造骨細胞增生和分化的影響

雙磷酸鹽藥物主要的藥理作用為抑制蝕骨細胞的骨吸收作用，因此被大量的運用在治療骨質疏鬆症和預防癌症的骨轉移。然而，在長期使用雙磷酸鹽藥物的病患中，卻發現部分病患在拔牙過後，竟會造成顎骨壞死的副作用現象(bisphosphonate-related osteonecrosis of the jaw, BRONJ)。部分假說認為顎骨壞死可能是由於雙磷酸鹽藥物降低蝕骨細胞的活性，進而降低骨頭的代謝(turnover rate)，因此產生BRONJ現象；另有假說認為，累積在骨頭的雙磷酸鹽濃度對於口腔上皮組織是有毒性的，導致軟組織傷口的癒合不完全。但BRONJ實際的致病機制還未明瞭。我們實驗室選用斑馬魚作為模式動物，以其尾鰭再生之特性，切除部分尾鰭模擬傷口的形成，探討在再生組織中，雙磷酸鹽藥物alendronate對於骨骼及上皮等組織的影響，希望能利用in vivo的研究來證實BRONJ的致病機制。之前本實驗室在形態學上的觀察中，發現雙磷酸鹽藥物alendronate對於成魚尾鰭再生有dose-dependent的影響，低濃度的雙磷酸鹽能促進斑馬魚尾鰭再生時骨頭生長；而高濃度的雙磷酸鹽則能抑制生長。另外，也發現alendronate會在斑馬魚尾鰭再生處造成細胞死亡的現象(吳俊學，2009)。因此，我以斑馬魚為實驗動物，將斑馬魚的尾鰭進行部分切除後，浸泡於低濃度(2.5×10-5M)及高濃度(7.5×10-5M)之alendronate中，觀察不同濃度之alendronate對於斑馬魚尾鰭再生組織之影響。實驗設計分為三部分，首先，以DNA鹼基類似物EdU (5-ethynyl-2'-deoxyuridine)浸泡於上述以alendronate藥物作用之斑馬魚，再由帶有螢光團的特定小分子，與EdU專一性結合後，可藉著螢光表現得知再生組織區域細胞增生的情形。再來，以綠色螢光蛋白標定造骨細胞的轉殖斑馬魚(Osterix promoter-GFP)，切除魚鰭後浸泡於不同濃度之alendronte藥物中，觀察在不同天數中(第3、5、7、9及11天)，綠色螢光表現之變化，得知造骨細胞在再生區域受到alendronte藥物作用的情形。最後，利用原位雜合反應，觀察斑馬魚魚鰭再生組織中，Runx2b、Runx2a及Collagen 10a1基因的表現，得知不同濃度的alendronate對於造骨細胞分化時造成的影響。實驗結果顯示，alendronate在in vivo中會對細胞增生有dose-dependent 的影響：低濃度alendronate能促進前期(截尾第三天時)(3 dpa, day-post-amputation)再生組織中細胞的增生，進而促使之後骨頭的再生；而高濃度alendronate則會抑制前期再生組織中細胞的增生，進而抑制之後骨頭的再生。另外，以綠色螢光蛋白標定造骨細胞的轉殖斑馬魚(Osterix promoter-GFP)觀察中發現，在造骨細胞中，高濃度alendronate會抑制再生組織中造骨細胞增生，促進造骨細胞死亡，影響骨生成作用。進一步利用原位雜合反應觀察斑馬魚魚鰭再生組織中，Runx2b、Runx2a及Collagen 10a1基因表現，結果發現alendronae並不會明顯地影響造骨細胞的分化，所以不是藉由影響造骨細胞的分化而影響骨生成作用。由以上結果推得結論，造成BRONJ的原因，不僅僅只是雙磷酸鹽抑制蝕骨細胞活性，降低骨頭的turnover rate；在活體實驗中發現，高濃度雙磷酸鹽藥物也會對造骨細胞產生影響，抑制造骨細胞的增生而非影響其分化，影響正常的骨生成作用。

關鍵字

骨壞死；雙磷酸鹽；斑馬魚；造骨細胞

並列摘要

The main mechanism of bisphosphonate on bone is to inhibit osteoclast-related bone resorption. Bisphosphonates have been prevalently applied in treatment of osteoporosis and prevention of bone metastasis. However, after tooth extraction, the patients who have taken long-term bisphosphonate treatment will appear the osteonecrosis of jaw (bisphosphonate-related osteonecrosis of the jaw, BRONJ). Some studies suggest that bisphosphnates impede the activity of osteoclast, then decrease the bone turnover rate, and finally lead to osteonecrosis of the jaw bone. Other studies also indicate that accumulated bisphosphonates are toxic to epithelium cell, resulting in incomplete soft tissue wound healing. However, the in vivo mechanism of BRONJ is still elusive. We choose zebrafish as the animal model, amputate the zebrafish caudal fin, and then treat with different concentration alendronate. Finally, we explore the effects of alendronate in the regeneration tissue to uncover the in vivo mechanism of BRONJ. Our previous studies have shown that alendronate is dose-dependent in regeneration of zebrafish caudal fin：low concentration of alendronate promotes bone regeneration, while high concentration of alendronate inhibits bone regeneration. Moreover, alendronate can also induce cell apoptosis in regeneration tissue of zebrafish caudal fin (Wu, 2009). In this study, I amputate the zebrafish caudal fin, and then treat them with low and high concentrations of alendronate (2.5×10-5M and 7.5×10-5M). The experiments are divided into three parts. First, DNA analog Edu (5-ethynyl-2'-deoxyuridine), which can bind with specific flourescent molecule (Alexa488), mark the cell proliferation site. EdU labeling revealed the cell proliferation in regeneration tissue. Second, I use the transgenic zebrafish (Osterix promoter-GFP) to observe the GFP expression in osteoblast under the treatment of alendronate. Finally, I perform in situ hybridization to detect early and late gene markers of osteoblast to unveil the gene expression patterns. My results show that alendronate has a dose-dependent effect in cell proliferation. Low concentration of alendronate increase cell proliferation, and further promote bone regeneration; high concentration of alendronte decrease cell proliferation, further inhibit bone regeneration. Moreover, by observing GFP-labeled osteoblast in transgenic zebrafish, I found that high concentration alendronate can inhibit osteoblast proliferation in regeneration tissue and induce osteoblast apoptosis. Furthermore, in situ hybridization experiments have demonstrated that alendronte doesn’t affect the differentiation of ostoblast. In conclusion, the studies revealed that alendronate inhibits proliferation and survival of osteoblast, but not differentiation, and leads to abnormality and retardation of bone regeneration.

並列關鍵字

bisphosphonate ； osteonecrosis ； zebrafish ； osteoblast

參考文獻

Mariotti, A., 2008. Bisphosphonates and osteonecrosis of the jaws. Journal of dental education 72, 919-929.

Akimenko, M.A., Mari-Beffa, M., Becerra, J., Geraudie, J., 2003. Old questions, new tools, and some answers to the mystery of fin regeneration. Developmental dynamics : an official publication of the American Association of Anatomists 226, 190-201.

Barrera, P., Blom, A., van Lent, P.L., van Bloois, L., Beijnen, J.H., van Rooijen, N., de Waal Malefijt, M.C., van de Putte, L.B., Storm, G., van den Berg, W.B., 2000. Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis. Arthritis and rheumatism 43, 1951-1959.

Bayliss, P.E., Bellavance, K.L., Whitehead, G.G., Abrams, J.M., Aegerter, S., Robbins, H.S., Cowan, D.B., Keating, M.T., O'Reilly, T., Wood, J.M., Roberts, T.M., Chan, J., 2006. Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish. Nature chemical biology 2, 265-273.

Bijvoet, O.L., Nollen, A.J., Slooff, T.J., Feith, R., 1974. Effect of a diphosphonate on para-articular ossification after total hip replacement. Acta orthopaedica Scandinavica 45, 926-934.

國際替代計量

利用斑馬魚尾鰭再生探討雙磷酸鹽藥物造成顎骨壞死的致病機制-造骨細胞增生和分化的影響

全文下載

主題瀏覽