臺灣博碩士論文加值系統

近十到二十年來，雙磷酸鹽藥物的使用已是處理骨流失相關疾病如骨質疏鬆症、柏哲德氏症(Paget''s Disease)、多發性骨髓瘤(multiple myeloma)及癌症骨轉移等病變的標準治療用藥，來防止疾病造成的骨溶性病徵、疼痛及骨折。
然而，此藥物也對牙科治療帶來新的衝擊。近年來日漸增多的雙磷酸鹽顎骨壞死(bisphosphonate-related osteonecrosis of the jaw, BRONJ)使牙科處理拔牙、牙周手術及植牙等與顎骨相關的治療內容必須格外小心。為了進一步理解BRONJ之成因，許多學者已由細胞實驗的成果得知蝕骨細胞最容易受雙磷酸鹽的影響而凋亡，因此後續跟進的實驗多針對雙磷酸鹽造成蝕骨細胞生理及訊息傳遞途徑的改變來設計實驗。目前已有相當豐碩的細胞學研究成果及臨床追蹤統計資料，然而，對於徹底解開BRONJ的成因仍沒有直接的證據。
本實驗使用斑馬魚尾鰭的硬骨作為研究BRONJ的動物模式，並利用抗酒石酸磷酸酶(Tartrate-resistant acid phosphatase, TRAP)的特性作為呈色的方法，標定活體內的蝕骨細胞，藉此觀察在Alendronate這種雙磷酸鹽的影響下，蝕骨細胞受到的影響。
實驗的第一部分使用TRAP呈色法，觀察斑馬魚截切尾鰭後24小時內，蝕骨細胞之活化時間、移動方向的動態變化。
實驗的第二部分則是將斑馬魚截切尾鰭後分成四組，分別在一般養魚用水(control, 0M Alendronate)及2.5×10-5 M、5.0×10-5 M及7.5×10-5 M 的Alendronate中飼養，來觀察截切尾鰭後第一、三、五、七、九及十一天的尾鰭再生長度及情況，並利用TRAP呈色法，觀察尾鰭上之蝕骨細胞位置及數量變化，藉以釐清活體魚之蝕骨細胞及尾鰭鰭條骨再生如何受雙磷酸鹽的影響。
24小時內的呈色結果顯示TRAP+的細胞來源可能源自血液循環系統的前趨細胞(precursor cells)，而非鰭條骨中間的間質細胞所延伸分化出來。十一天的尾鰭再生觀察發現TRAP的呈色訊號隨含氮雙磷酸鹽的濃度上升而減少，且訊號出現的時間點延後，出現在鰭條上的時間也縮短。可間接證明文獻提到雙磷酸鹽抑制蝕骨細胞表現TRAP，甚至影響其活性。尾鰭再生長度隨雙磷酸鹽濃度升高而縮短，顯示雙磷酸鹽雖然抑制蝕骨細胞活性，但尾鰭與鰭條骨骼再生也受抑制，相較於先前細胞實驗的結果，此活體實驗顯示尾鰭與鰭條骨再生過程可能有更複雜的細胞交互作用存在，從而導致BRONJ的形成。由此也反映出BRONJ之病因學理上細胞交互作用的複雜性。

Bisphosphonates have been predominately applied in the treatment of osteolytic bone diseases, such as osteoporosis, Paget''s disease, multiple myeloma, and cancer-related metastatic bone lesions for the past several decades. Their clinical applications have alleviated the osteolytic symptoms, pain and fractures entailed by those diseases.

However, the implications of those drugs have led to new impacts on clinical dental treatments. Recently, an increasing number of clinical cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) have been reported, rendering the precautious measures in the treatments of dental extractions, periodontal surgery and dental implants necessary. Previous studies have shown that osteoclasts are the primary target cells and most vulnerable to bisphosphonates treatments. However, most of the experiments were confined to in vitro cell culture studies, simplicity of cell types in the experiments. On the other hand, the in vivo studies under physiological conditions are still elusive. Although clinical statistics and research in the cell biology field have gained some insights and have led to several hypotheses, the etiology of BRONJ remains to be explored in much more details.

To investigate the effects of bisphosphonate on osteoclasts and to unravel the in vivo mechanism underlying BRONJ, I employed a fin-regeneration model in an animal model--zebrafish. Zebrafish caudal fin rays were amputated and allowed to regenerate in the presence of alendronate, an aminobisphosphonate, at three doses. The growth tendencies of the fin ray bone were observed for 11 days after amputation. Osteoclasts were located by TRAP staining during the regeneration process.

In the first part of my study, I observe the dynamic appearance of osteoclasts after fin-amputation within 24 hours by TRAP staining without any drug treatment.

In the second part of the experiments, to clarify the impact of bisphosphonates on osteoclasts during the process of fin regeneration, I investigate the long-term effects of alendronate on osteoclast’s survival and distribution using the TRAP staining. Zebrafish were treated under three doses of administration of alendronate, 2.5×10-5 M, 5.0×10-5 M, 7.5×10-5 M, and 0 M as control. The samples were collected, stained, and observed at the first, 3rd, 5th, 7th, 9th and 11th day after amputation.

The results within 24 hours showed that by 18 hr, very few osteoclasts were scattered in the fin. In contrast, after 18 hr, many osteoclasts gradually gathered in the vicinity of amputated border sites evenly, located in the newly formed tissues as well as in the original tissues. After amputation, the dynamic distribution of osteoclasts within 24 hr implies that those cells may come from the circulation system and invade into the amputated border sites via extravasation, rather than from the local mesenchymal cells between fin hemirays.

The results of the long-term observation showed that the distribution of osteoclasts gradually diminished under the influence of alendronate administration; the appearance of TRAP+ osteoclasts was retarded and the duration of those cells on the regenerated fin was shortened. Those observations suggest that the survival and cellular activity of osteoclasts were impaired by alendronate. Contentiously, although the distribution and activity of osteoclasts were impeded by alendronate administration, the regeneration of fin ray (bone) was not ameliorated at high doses of alendronate administration. In contrast to previous studies in cell culture system, the results indicate that promiscuous cellular interaction exist during bone repairing, reflecting the etiological complication of BRONJ.

口試委員會審定書 I
誌謝 II
中文摘要： III
英文摘要： V
壹、引言 1
貳、文獻回顧 3
一、雙磷酸鹽相關的顎骨壞死(BISPHOSPHONATE-RELATED OSTEONECROSIS OF THE JAW, BRONJ) 3
二、蝕骨細胞特性與蝕骨細胞新生(OSTEOCLAST AND OSTEOCLASTOGENESIS) 10
三、抗酒石酸磷酸酶(TARTRATE-RESISTANT ACID PHOSPHATASE)的生物化學性質 23
四、TRAP基因表現的控制 25
五、蝕骨細胞TRAP酵素的骨吸收功能 28
六、TRAP在骨吸收作用中的機制 29
七、研究動機 32
參、實驗材料 35
肆、實驗方法 39
一、利用TRAP染色觀察斑馬魚(ADULT FISH)截尾後蝕骨細胞活化、移動、及分布狀況 39
二、斑馬魚(ADULT FISH)浸泡ALENDRONATE溶液並利用TRAP染色方式來觀察尾鰭再生及蝕骨細胞分布情形 41
伍、結果 43
一、24小時內，對照組之蝕骨細胞TRAP酵素呈色反應 43
二、依濃度分組，長時間(1～11天(D)，1～11 DAY-POST-AMPUTATION, DPA)觀察浸泡ALENDRONATE的斑馬魚尾鰭再生時TRAP呈色位置及分布。 44
陸、討論 48
一、TRAP +的蝕骨細胞受雙磷酸鹽的影響而使存在的數量下降 48
二、表現TRAP+的細胞可能包含有蝕骨細胞以外的其他細胞來源 48
三、魚隻個體差異是非常明顯的影響因素 50
四、TRAP染點位置出現在截切處的近心端 50
柒、結論 52
捌、圖 53
圖一、 53
圖二、 54
圖三、 55
圖四、 56
圖五、 57
圖六、 58
圖七、 59
圖八、 60
玖、參考文獻 61

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