以基因轉殖方法及原位雜合反應探討ctsk基因在蝕骨細胞於斑馬魚發育及尾鰭再生過程之表現及其受雙磷酸鹽作用之影響

Abstract

蝕骨細胞是一種能夠吸收骨基質的細胞，為單核細胞經由細胞融合而來。蝕骨細胞和造骨細胞能夠互相配合並產生交互作用，一加一減地調節骨頭的生長及恆定以及控制骨頭的外形。蝕骨細胞要進行骨吸收時，會緊貼於骨頭表面，並且在氫離子幫浦的幫助下，營造出一種酸性的環境，進而活化部分溶小體酵素，例如tartrate resistant acid phosphatase (TRAP)、cysteine proteases（例如 cathepsin K, (CATK, CTSK)）、carbonic anhydrase II等，分泌到細胞外，而達到骨質吸收的目的。Cathepsin K為一種能夠辨認半胱氨酸(Cysteine)的蛋白質分解酶，其受質包括第一型膠原蛋白和黏骨素 (osteonectin)。 而cathepsin K是屬於c1蛋白家族中的成員，主要在蝕骨細胞中表現。 為了進一步探討蝕骨細胞在生物體發育過程中所扮演的角色，我以斑馬魚為模式動物，分別將人類以及斑馬魚ctsk基因的啟動子結合βB1-Crystallin基因1.3kb的加強子片段及100bp的啟動子片段合併再與綠色螢光蛋白報導基因接合製成結構體，在斑馬魚受精卵一個細胞期時，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，藉由觀察螢光的表現，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位置。 共篩選到六個轉殖恆定品系(transgenic stable line)；三個來自人類CTSK啟動子；另外三個來自斑馬魚ctsk啟動子。結果發現，經人類CTSK基因啟動子所驅動螢光表現的轉殖斑馬魚在一至二周大時，螢光只表現在眼部，而當三至四周時，可在眼睛水晶體、鰓蓋骨，以及鰓蓋後下方骨骼，腹鰭基底端發現螢光。最後在成魚時，除了上述部位外，亦可於肋骨，尾鰭等處觀察到螢光表現。此外，經由魚類ctsk基因啟動子所驅動之轉殖斑馬魚的螢光表現較強、範圍較廣且出現時間較早；同樣表現在眼睛水晶體、鰓蓋骨，以及鰓蓋後下方骨骼，腹鰭基底端。但是在三個轉殖品系中卻無法在尾鰭鰭條上偵測到螢光表現。 為了探討CTSK基因於蝕骨細胞在骨再生作用 (bone regeneration) 時的表現情形，我進而進行轉殖斑馬魚的截尾實驗並且利用in situ hybridization的方式去做檢測及驗證。將成功轉殖人類ctsk啟動子的斑馬魚F1世代 (H5 line)施行尾鰭半截尾，去觀察尾鰭再生時螢光表現的動態變化。另一方面，野生型斑馬魚同樣也進行切尾，並於尾鰭再生時不同時間點再次收集標本，取得尾鰭新生組織，進行in situ hybridization。結果發現，尾鰭再生時，螢光蛋白的表現於再生第一天尾鰭截點處開始表現，在二至四天持續增強，並在截點至新生端點皆可觀察到螢光，第五天開始螢光表現亮度減弱，而至第九天時，螢光表現之亮度已無明顯增強的現象。然而在in situ hybridization這部分的實驗，設計引子去標定ctsk mRNA的表現位置，早期(2-3dpa)表現於截點後新生組織，晚期則發現只在尾鰭新生組織的端點處可偵測到訊號，同時也在再生後二至五天可發現訊號增強的現象。此兩組實驗在表現位置上結果的出入，推測是蛋白質與訊息核醣核酸表現時間點以及降解速度不一所致。 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劑量效應 (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，(吳俊學，2009)因此，我也進行雙磷酸鹽藥物 alendronate的泡藥實驗，希望能了解該藥物在尾鰭再生時對於蝕骨細胞表現的影響。不論是原位雜合反應偵測CTSK mRNA或是觀察轉殖品系報導基因GFP螢光表現之結果均顯示，ctsk基因的表現程度相較於控制組來得低，而且浸泡高濃度(7.5×10-5M)之實驗組更有生長及表現延遲的現象。顯示雙磷酸鹽藥物在尾鰭再生過程中，會抑制蝕骨細胞的活性及分化程度。

Osteoclasts, multi-nucleated cells involved in bone resorption, are derived from the monocyte-macrophage cell lineage via fusion of precursor cells. Along with osteoblasts, these cells are implicated in bone remodeling by degrading the organic matter and dissolving mineralized bone matrix. The interaction and balance between osteoclasts and osteoblasts orchestrate bone homeostasis; osteoblasts conduce to bone formation, while osteoclasts facilitate bone resorption. Several characteristic proteolytic enzymes are highly expressed in osteoclasts such as tartrate resistant acid phosphatase (TRAP), cysteine proteases and carbonic anhydrase II. Cathepsin K, a member of cysteine proteases, encoded by ctsk gene, is expressed predominantly in osteoclasts. It belongs to the peptidase C1 protein family and is a lysosomal cysteine protease implicated in bone remodeling. To explore the functional role of osteoclates during ontogeny and bone regeneration, I use zebrafish as a model animal and create transgenic stable lines. I utilize CTSK promoter and green fluorescent protein reporter gene (GFP) to label osteoclasts and track their development. To this aim, first I constructed plasmid constructs that combines the promoter of human or zebrafish ctsk gene with βB1-Crystallin 1.3kb (Cr1.3) enhancer elements and βB1-Crystallin 100bp promoter (Cr basal promoter), upstream of EGFP reporter gene. Then, these constructs were microinjected into one-cell stage zebrafish embryos. The expressions of EGFP were observed. In this study, I profit theβB1-Crystallin 1.3kb (Cr1.3) enhancer elements and 100bp promoter, which confer tissue-specificity in the lens as a selection marker for screening transgenic zebrafish as early as 3~4 dpf (day-post fertilization). I harvested 6 transgenic stable lines, three from human CTSK promoter construct and three from zebrafish ctsk promoter construct. A transgenic line H5, which is driven by human ctsk promoter, showed fluorescence in the regions of lens, craniofacial bones, ribs, and tail fin rays. On the other hand, the three lines of the zebrafish ctsk promoter expressed GFP stronger than those of human promoter. However, these three lines of zebrafish ctsk promoter fail to express EGFP in tail fin rays, even during the fin ray bone regeneration. To monitor the expression of CTSK gene in osteoclasts during bone regeneration, I employed fin amputation-regeneration measures in the transgenic zebrafish line H5 to observe the dynamics of EGFP expression as indicator. In addition, I perform in situ hybridization to locate endogenous ctsk mRNA expression during fin regeneration as a reference. The results showed that both the fluorescence of GFP and ctsk mRNA expression culminated at 3 dpa (day-post-amputation), then started to fade away at 5 dpa, and were restored to the basal line at 9 dpa. Yet the expression regions of GFP and mRNA are differential; the EGFP is expressed along the fin rays evenly, while the mRNA is expressed in the new regenerated fin tissues (2-3 dpa) and later only in the marginal zones close to blastema. The disparity may be ascribed to the different timing of expression and degradation. Our previous studies have shown that alendronate is dose-dpendent in the regeneration of zebrafish caudal fin: low concentration of alendronate promotes bone regeneration, while high concentration of alendronate inhibits bone regeneration (Wu, 2009). To evaluate the influences of alendronate on osteoclasts during fin regeneration, I treated the amputated zebrafish at two concentrations of alendronate, detected the dynamic EGFP expression, and performing in situ hybridization of ctsk expression. The results demonstrated that the application of alendronate diminished the expression of ctsk gene and led to its retardation.