利用葉基香葉醇挽救斑馬魚尾鰭再生時因雙磷酸鹽損害細胞之功能

Abstract

雙磷酸鹽類藥物引起之顎骨壞死是雙磷酸鹽治療主要副作用之一。至今，尚未有對於BRONJ的有效治療方式。含氮雙磷酸鹽（N-BPS）抑制焦磷酸合酶（FPPS）在mevalonate pathway（MVP）。香葉基香葉醇（GGOH）和香葉醇(FOH)代謝產物的下降被認為是顎骨壞死的主要因素。先前的體外研究已經顯示含氮雙磷酸鹽(N-BP)中的負面影響，降低不同類型的細胞的生存力和遷移的能力。本研究的目的是要測試，下游代謝產物的應用可以逆轉含氮雙磷酸鹽中負面的生物學效應。

我們利用斑馬魚的鰭再生模型探討其影響，試圖以mevalonate pathway下游生化產物在野生種(wild type) 斑馬魚中進行挽救實驗(rescue experiment)，進行更細微之觀察及研究。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，藉此觀察在香葉基香葉醇(geranylgeraniol, GGOH) 和香葉醇(FOH)的影響下，蝕骨細胞受到的影響。

本實驗首先利用calcein活體染色來觀察此藥物對成魚尾鰭切除後再生之型態是否有影響。再利用TRAP呈色法，觀察斑馬魚截切尾鰭後，蝕骨細胞之活化時間、移動方向的動態變化。藉以釐清活體魚之蝕骨細胞及尾鰭鰭條骨再生如何受香葉基香葉醇的影響。

實驗結果顯示，尾鰭再生觀察發現TRAP的呈色，雙磷酸鹽會抑制蝕骨細胞表現，甚至影響其活性。而香葉基香葉醇(geranylgeraniol, GGOH) 和香葉醇(FOH)對尾鰭再生部分發現骨質再生具有顯著影響，具有促進骨質生成作用。其結果可進一步延伸到在治療BRONJ的臨床應用。本次研究發現geranylgeraniol(GGOH)的調節蝕骨細胞的處理，更凸顯了GGOH的重要性，其臨床應用是進一步研究的課題。

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is one of the main side effects of bisphosphonate therapy (BPT). To date, there is no effective therapy of the BRONJ. Nitrogen-containing bisphosphonates (N-BPs) target to the inhibition of pyrophosphate synthase (FPPS) in the mevalonate pathway. Consequently, decreased synthesis of the downstream metabolites, geranylgeraniol (GGOH) and farnesol (FOH), is believed to largely account for the development of BRONJ. Previous in vitro studies have shown the negative effects of N-BPs on decreased viability and migration capacity of different cell types. The aim of our study was to demonstrate that the application of downstream metabolites may reverse the negative biological effects of N-BPs. Amputation-regeneration model of zebrafish caudal fin was employed to analyze the effects of drug administration. Alendronate (N-BP), farnesol, and geranylgeraniol at 7.5×10-5M were used to treat the fish by incubation. The regenerated bones in the fin were documented by calcein staining. The dynamic appearance of ostelclasts during the process was observed by TRAP staining. The bone regeneration impaired by alendronate was reversed to normal in the presence of GGOH and FOH. In addition, the morphological restoration was delineated by TRAP staining to reveal the distribution of osteoclasts. The results showed that the number and distribution of osteoclasts were restored to normal likewise. Hence, systemic application of GGOH and FOH may reduce the side effects of bisphosphonate therapy. In this study, we demonstrate that the impairing effects of alendronate on bone regeneration and osteoclast activities may be reversed by application of GGOH and FOH. Our study in zebrafish provides a vivid animal model to investigate the BRONJ in vivo. And the results may further extend to the clinical applications in treating BRONJ.