蝕骨細胞特異性基因啟動子Acp5b 轉殖實驗及觀察在發育過程蝕骨細胞的分佈

Transgenic Assay of Osteoclast-Specific Acp5b Gene Promoter and Observing the Distribution of Osteoclasts

林瑜均(Yu-Chun Lin)

指導教授 ： 張百恩

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603338

摘要

當骨頭受到周遭機械壓力或荷爾蒙影響，會進行骨頭的重建修復作用，骨頭進行重建時會包含骨質的破壞吸收和骨頭的再生。當骨頭要進行重建時，骨表面的造骨細胞釋放出一些化學訊號因子促使蝕骨細胞遷移到需要重建骨質的地方，形成多核的成熟蝕骨細胞。接著成熟的蝕骨細胞進行骨質溶蝕作用，此時蝕骨細胞釋放溶小體內的酵素，這些酵素於酸性環境才有活性，氫離子幫浦會釋出氫離子，使外在環境處於酸性狀態，釋出鈣離子與磷酸根離子。分解之後，造骨細胞再過來進行骨頭重建再生。所以蝕骨細胞與造骨細胞在骨吸收和骨形成之間的耦合過程中扮演十分重要的角色。蝕骨細胞進行溶蝕時所釋放出的酵素包含tartrate resistant acid phosphatase (TRAP)、cysteine proteases (例如 cathepsin K）等。其中抗酒石酸磷酸酶 (Tartrate- resistant acid phosphatase, TRAP) 普遍存在於蝕骨細胞，可以作為蝕骨細胞的標記物。

所以我的目標是進行蝕骨細胞特異性基因Acp5b (=TRAP)啟動子(promoter)的轉殖實驗，並用紅色及綠色螢光標記，遠程目標可以進一步利用細胞毒殺的方式毒殺這些蝕骨細胞並觀察骨頭再生的情形。實驗以斑馬魚為模式生物，將斑馬魚Acp5b基因的啟動子1.2kb片段與綠色及紅色兩種螢光蛋白報導基因結合形成結構體，在斑馬魚受精卵約十幾分鐘一個細胞期時(15-30 min)，以顯微注射的方式注入斑馬魚受精卵中，期望能夠在斑馬魚轉殖實驗中，藉由觀察紅色綠色螢光的表現及分佈位置，而得知蝕骨細胞在斑馬魚發育過程中的表現情形以及分布。
實驗結果顯示，在斑馬魚眼睛及骨頭分佈看得到螢光的訊號，成功進行acp5b的cloning，並得到多組F2的stable line。蝕骨細胞大致上分布在頭骨、鰓蓋、尾鰭、背鰭、臀鰭以及脊椎骨上下緣。大約在三天可以看到魚眼睛的螢光訊號，約五天會發現小魚的腎臟也開始出現蝕骨細胞轉殖螢光訊號，一週可發現頭部開始產生蝕骨細胞螢光訊號，10天尾柄處也開始看得到螢光訊號。到了兩週開始，及椎骨上下緣出現螢光表現， 3週可發現胸鰭、背鰭、臀鰭長出後也產生螢光訊號。接著等斑馬魚性成熟(約兩個月大)進行截尾觀察尾鰭再生的蝕骨細胞分佈，但我們在截尾這部份未發現蝕骨細胞acp5b的螢光訊號，推測控制大魚尾鰭再生的增強子可能位於另一個控制斑馬魚TRAP的基因acp5a上面。這個實驗記錄斑馬魚生長階段蝕骨細胞的分佈，並得知尾鰭骨頭再生過程acp5b不會表現。

在後續遠程實驗的部分我們可用細胞毒殺的方式觀察蝕骨細胞毒殺後骨頭的生長分布，毒殺細胞所需的reductase還原酶的cDNA已經跟Dr.Peter F.Searle實驗室取得，之後可以進一步作出這些還原酶的construct，再利用我們的acp5b啟動子啟動這些帶有還原酶的construct，以細胞毒殺的方式毒殺這些蝕骨細胞並觀察骨頭再生的情形。

關鍵字

蝕骨細胞 ； TRAP(ACP5) ； 骨再生作用 ； 斑馬魚 ； 發育

並列摘要

Bone remodeling will occur to rebuild and repair bone when it encounter mechanical stress or hormonal influence. The procedures of bone remodeling involve bone destruction, absorption, and bone regeneration. During bone remodeling, osteoblasts on the bone surface will release some signal factors for promoting osteoclasts to migrate to the site where needed to be re-built, and forming mature multinucleated osteoclasts. Matured osteoclasts dissolve bone by secreting acid and enzymes that degrade the organic bone matrix in this acidic environment. Before osteoclasts break down, osteoblasts replace them when receiving signals and produce new bon matrix. So the coupling between osteoclasts and osteoblasts play a cruial role in the process of bone resorption and formation. For the purpose of degrading bone matrix, absorbing osteoclasts release some enzymes like tartrate resistant acid phosphatase (TRAP), cysteine proteases (for example, cathepsin K) and so on. Among them, TRAP, abundant in osteoclasts, can be used as an osteoclast marker.

My research goal is to do transgenic assay of osteoclast-specific Acp5b (= TRAP) gene promoter, using red and green fluorescent proteins as reporter genes to label osteoclasts and to track their development. To this aim, I use zebrafish as a model animal and make a construct harboring 1.3kb zebrafish Acp5b promoter and red- and green-fluorescent protein reporter genes to label osteoclasts. Then, these constructs were microinjected into one-cell stage of zebrafish embryos. The expressions of EGFP and DsRed were observed.

My results reveal that the fluorescence signals are visible in zebrafish eye and around the bones in transient assays (F0). Then I screened F0 and obtained F1 stable lines. I got 17 stable lines in which DsRed can be observed in the eyes. However, only two lines also express DsRed reporter gene in osteoclasts. I found that DsRed-labelled osteoclasts were broadly distributed in the skull, gill cover, pectoral fin, tail fin, dorsal fin, anal fin and upper and lower edges of dorsal and ventral spines.

In addition, to monitor the expression of Acp5b gene in osteoclasts during fin regeneration, I employed fin amputation-regeneration measures in the stable zebrafish line F1 (No. 7 and 16, about 2 month dpf). However, fluorescent signals were not detected in osteoclasts after amputation. Presumably, the enhancers to control TRAP expression in zebrafish osteoclasts during tail fin regeneration may be located in another TRAP gene, Acp5a.

My experiments record the distribution of zebrafish osteoclasts in early developmental stages. In the future, we can apply cytotoxicity using a bacterial nitroreductase to convert a prodrug, Metronidazole, into a cytotoxic metabolite to ablate osteoclasts and observe the bone morphology in the absence of osteoclasts during zebrafish bone growth. By this way, we can evaluate the cell function of osteoclast during bone growth and regeneration

並列關鍵字

osteoclast ； acp5b ； zebrafish ； bone regeneration ； development

參考文獻

Adams, L. M., M. J. Warburton and A. R. Hayman (2007). "Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP)." Cell Biol Int 31(2): 191-195.

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