以斑馬魚模式動物探討雙磷酸鹽造成蝕骨細胞分化與分布的影響

Abstract

目的： 雙磷酸鹽類藥物於1980年代開始出現於市面上，在臨床上常應用於骨質疏鬆症、惡性腫瘤全身性骨轉移、多發性骨髓瘤等病症上。截至2006年的統計，美國有超過兩千四百萬個處方籤被開出，全球則是開出近兩億個口服處方籤，而且有將近三百萬個癌症病人接受雙磷酸鹽靜脈注射，可見其使用的廣泛性(Uyanne et al., 2014)。然而在藥物上市一段時間後，顎骨壞死的副作用陸續出現。最早是在2003年由美國口腔顎面外科醫師 Marx提出36個案例報告，之後陸續學者統計有上萬例的報告，這也使得醫界對於此藥物副作用有警覺心。 由於此藥物會影響到顎骨傷口的癒合，凡是有關牙科的手術範圍，小至拔牙、牙結石刮除，大到牙周手術、植牙，都要特別注意。一旦發生了顎骨壞死的情形，那麼等待傷口癒合及觀察的時間就會需要很久，術後的重建方式也是相當令人頭痛，通常也只能做消極或非侵入性的處理。因此，我們期望可以了解雙磷酸鹽相關顎骨壞死(BRONJ)的成因，以期可以找出可以預防的評估與治療的方法。 近年來有多篇研究有對於BRONJ提出可能的致病機轉，一般認為主要是會抑制蝕骨細胞的作用，進而導致骨修復功能缺損，但無法得知其直接的作用機制。因此，我的研究目標是想探討雙磷酸鹽類藥物對於蝕骨細胞分化及分布的影響。 實驗材料與方法： 由於斑馬魚的尾鰭再生與人類膜內骨再生方式類似，因此本實驗以斑馬魚的尾鰭再生作為研究BRONJ的動物模式，來觀察雙磷酸鹽對於蝕骨細胞作用機制的影響。我們一共分成兩組實驗魚群，其中一組是野生組(wide type)，會利用抗酒石酸磷酸酶(Tartrate-resistant acid phosphatase, TRAP)呈色方法來標定活體內的蝕骨細胞；另一組則是選用經人類CTSK 基因啟動子，標定蝕骨細胞的基因轉殖斑馬魚，希望可以藉此觀察雙磷酸鹽對蝕骨細胞的影響。 首先將斑馬魚尾鰭切除後，每個組別分別養在飼育過濾水(對照組)、低濃度(2.5×10 -5M)及高濃度(7.5×10 -5M)的Alendronate中，在截尾後的第三天及第五天時，我們將再生成的尾鰭再次切除，利用TRAP染色，並將這些標本做樹脂包埋，然後切片做尾鰭橫截面的組織學觀察，如蝕骨細胞的細胞型態、以及在再生尾鰭處分布的情形。觀察蝕骨細胞的細胞形態，可以推論其分化及功能受到的影響，進而比較藥物濃度和蝕骨細胞被抑制程度的相關性。另外，在橫截面上觀察由TRAP標定蝕骨細胞的分布位置，也可以有助於我們了解、推論藥物對骨吸收作用的影響。 最後，將TRAP呈色組，和CTSK基因轉殖組所標定出來的蝕骨細胞做比較，觀察其分布位置上表現的異同，以推論兩者所標定到蝕骨細胞的來源或時期是否相同。 結果： 實驗結果顯示，在有浸泡雙磷酸鹽的組別，蝕骨細胞的型態較不易平貼鰭條骨表面，顯示其分化受到抑制，行使骨吸收的功能受損。另外，蝕骨細胞的TRAP表現以及尾鰭再生長度都會隨著雙磷酸鹽的濃度增加而減少，我們推測是因為雙磷酸鹽藥物會造成蝕骨細胞的功能抑制甚至凋亡，而導致尾鰭再生及修復功能的異常。 TRAP的蝕骨細胞分布，在靠近截尾處及再生尾鰭末端比較明顯，數量也較多，而CTSK的蝕骨細胞相較之下數量較少但分布的較均勻。由以上結果推測，TRAP的蝕骨細胞來源可能源自血液循環系統的前驅細胞(Precursor cells)，而CTSK標定出的蝕骨細胞來源，可能是由鰭條骨中間的間質細胞所延伸出來。 總結： 此實驗顯示在雙磷酸鹽的影響之下，蝕骨細胞的分化會受到抑制，進而影響到傷口的癒合、骨頭修復與再生的能力。因此臨床上常見服用此藥物的病人，會有BRONJ的情形產生。

Objectives： Bisphosphonates began to be prescribed in the 1980s, often used in osteoporosis, bone metastasis of malignant tumors, multiple myeloma. Over 200 million oral and nearly three million intravenous prescriptions were made until 2006. However, 36 cases of osteonecrosis of the jaw was first reported in 2003 by the American Oral and Maxillofacial Surgeons, Marx. Ten thousands cases was proposed later. People started to pay attention to the side effect of this drug. Because this drug delayed wound healing of the jaw bone, all dentoalveolar surgeries were involved, such as tooth extraction, root planing, periodontal surgery, or implant. Dentists pay special attention to this topic and phenomenon. Upon osteonecrosis of the jaw occuring, it takes long time to wait for wound healing. Moreover, reconstruction surgery is difficult and non-invasive treatment option was usually chosen. Thus, we would like to unveil the etiology of BRONJ, and hope to find the preventive evaluation and treatment methods. Various causative pathologies of bisphosphonate-related osteonecrosis of the jaw have been proposed. Inhibition of osteoclast function is generally considered to be the main mechanism, leading to cell apoptosis and defect in bone remodeling, but direct mechanism of action was unknown. Therefore, my research goal was to explore the effects of bisphosphonate on differentiation and distribution of osteoclast. Materials and Methods： Owing to the similar process between fin regeneration of zebrafish and intramembranous ossification bone growth of humans, we use zebrafish as an animal model to explore the effects of bisphosphonates on osteoctlast. Zebrafish were selected from two groups, one wild type group was revealed by TRAP staining (Tartrate-resistant acid phosphatase, TRAP) to mark osteoclasts; the other group is CTSK transgenic zebrafish by human osteoclast gene promoter encoding green fluorescent protein(GFP). The stain and fluorescence help us to observe the effect of bisphosphonates on osteoclasts in these fish. First of all, we amputate the zebrafish tail fin in half, and all fishes were divided into three groups according to breeding condition by concentration of Alendronate: the control group, the low concentration (2.5 × 10-5M) and the high concentration (7.5 × 10-5M) of Alendronate. On day 3 and day 5 after fin amputation, we collected the regenerated fin and revealed with TRAP staining. These specimens were fixed overnight at 4 °C in 4% paraformaldehyde (PFA), dehydrated by ethanol, and embedded in resin (Technovit 9100, Kulzer Heraeus) according to the manufacturer's instructions. Sections were cut at 7 μm and toluidine blue as counter stain. Then we observe the histology by cross-section slide, including the morphology of osteoclasts, the distribution of osteoclasts, to see if their differentiation or bone resorptive ability were suppressed by bisphosphonates. Also, we compared the extent of suppression by different drug concentration. Furthermore, these findings could help us understand the inhibited osteoclasts function affected by bisphosphonates. Finally, we compare the group of TRAP and CTSK transgenic zebrafish from gross view, expecting to find the similarities and differences of the distribution and origin of osteoclasts. Results： The results showed that multinucleated osteoclasts with rounded morphology increased after bisphosphonate use, their differentiation might be inhibited and resulted in impaired bone resorption. In addition, the TRAP stain of osteoclasts showed and the length of the regenerated tail were both decreased with increasing drug concentration, suggesting that bisphosphonates could cause the apoptosis of osteoclasts, supressed regeneration, and delayed wound healing. TRAP-marked osteoclasts were larger in number and distributed mostly at the cutting line and the area nearby the end of regenerated fin. Whereas the CTSK-specific osteoclasts were less revealed by TRAP stain and evenly-distributed over the regenerated fin. These results indicated that the origin of TRAP-marked osteoclasts was possible from the precursor cells in blood circulatory system, while CTSK-marked osteoclasts were probably derived from the mesenchymal cells of the fin rays. Conclusion： This experiment showed that osteoclast differentiation may be suppressed under the use of bisphosphonates, leading to increased cell apoptosis. Defects in wound healing, bone repair and regeneration may result in BRONJ.