選殖及分析斑馬魚蝕骨細胞專一性表現之ACP5a基因啟動子

Abstract

蝕骨細胞(osteoclast)做為一個吸收骨基質的細胞，在各種具有骨骼的生物中扮演重要角色，其起源是由骨髓中分化的單核細胞，經由訊息傳遞活化並產生細胞融合而形成，其外觀具有明顯的多細胞核構造與富含粒線體。蝕骨細胞和造骨細胞(osteoblast)藉由兩者間的訊息傳遞相互調節彼此的生長，在骨骼的重塑、生長的調節及外觀形態的控制上擔任重責。當蝕骨細胞進行骨基質的吸收時，該細胞會以皺褶緣(ruffled border)貼附於目標表面，用來增加分解骨基質之速率，而後藉由質子幫浦(proton pump)使該環境處於pH值約5的酸性，進一步使蝕骨細胞中的酵素如抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase, TRAP)被大量釋出，鈣離子與磷酸根離子被蝕骨細胞運輸並釋放到血液裡，從而完成這個骨基質吸收過程。抗酒石酸酸性磷酸酶(TRAP，又名acid phosphatase 5，ACP5)做為常見的檢測蝕骨細胞功能的標記，其功能為在酸性環境下將目標之磷酸根解離出來，並在後續鈣離子的釋放中，隨著鈣離子一同被釋放進血液，因此經由檢測血液中的TRAP濃度可以得知蝕骨細胞的活性。 抗酒石酸酸性磷酸酶在人類的基因名稱如同其酵素名，同樣為Acp5，然而在斑馬魚中產生了分歧，分別名為acp5a及acp5b，兩者間的機制與功能目前尚未釐清，在此我使用了acp5a作為我研究的標的。而在acp5a中，又可細分出三個不同的轉錄起始點，分別位在Exon 1a，Exon 1b及Exon 1c上，因此在此篇論文中，將會探討這三個轉錄起始點與啟動子(promotor)片段活性。 在過去實驗室學長姊的研究中得知，在斑馬魚截尾再生的過程中，可以用TRAP染色染到抗酒石酸酸性磷酸酶。為了接續探討抗酒石酸酸性磷酸酶在骨吸收與再生中扮演的角色，這篇論文裡，我使用斑馬魚作為模式動物，並且利用Tol2的轉位子系統以及以紅色螢光蛋白(DsRed)做為報導基因，經由顯微注射的方式來建立一個帶有抗酒石酸酸性磷酸酶啟動子的品系。在過渡性實驗(F0)中，斑馬魚於孵化後7-10天利用螢光顯微鏡觀察時，除了明顯的眼睛螢光外，在三種不同啟動子片段的組別中，Exon(1a+1b)+ TATA276bp(Exon1c+2)的組別可明顯在全身脊椎發現節狀亮點，而在Exon(1c+2)組以及Exon(1a+1b)組則沒有發現到。在卵黃的情況中，Exon(1c+2)組與Exon(1a+1b)+TATA276bp(Exon1c+2)組都能在7天左右的斑馬魚卵黃發現螢光，Exon(1a+1b)組依然沒有發現到。而在後續經由篩選建立的恆定品系(F1)當中，Exon(1a+1b)+TATA276bp(Exon1c+2)的組別，共篩選到3種身體帶有螢光的品系(分別命名為2號、7-1號與7-2號)，在身體螢光表現較強的7-1號與7-2號中，其螢光表現與目前已知的TRAP染色結果大致相同，在孵化後14天起陸續於脊椎骨、魚鰭鰭條、上下顎骨表現出紅色螢光，對斑馬魚成魚的觀察中也能發現到其身體表面與鱗片上的紅色螢光。除此之外，進一步對斑馬魚成魚進行截尾實驗，以觀察其在骨再生中的表現，在再生的尾鰭中也可以觀測到紅色螢光有從截尾的傷口擴散到新生尾鰭的情況，與過去實驗室內學長對尾鰭再生進行的TRAP染色研究結果大致相同。

Osteoclasts, cells that absorb bone matrix, play an important role in various bone metabolism. They are formed by the differentiation of monocytes in the bone marrow, activation by signal transduction, and then maturation via cell fusion with multi-nuclear structure and rich in mitochondria. Interactions between osteoclasts and osteoblasts are essential for the remodeling, the growth-regulation, and the control of morphology of bones. When osteoclasts absorb the bone matrix, the cells attach to the target surface with a ruffled border to confine a region where the bone matrix is broken down. In an acidic environment at a pH of about 5 mediated by a proton pump, the enzymes in the osteoclast, such as tartrate-resistant acid phosphatase (TRAP), are released in large quantities, and calcium ions and phosphate ions are transported by the osteoclasts and released into the blood, completing the bone-matrix absorption process. TRAP, also known as acid phosphatase 5 (ACP5), is a common marker for detecting the function of osteoclasts. Its function is to dissociate the phosphate from the targets in an acidic environment and release it with calcium ions. As TRAPs are released into the blood together with calcium ions, the activity of the osteoclasts can be detected by the concentration of TRAP in the blood. In zebrafish, two acp5 genes, acp5a and acp5b, are located in the genome. The mechanism and function of the two genes are still elusive. Herein, I study the enhancer functionalities of acp5a. From NCBI and Ensembl bioinormatics, three transcriptional start sites, located on Exon 1a, Exon 1b and Exon 1c, are present in acp5a. The conserved enhancers and promoter regions around these three transcriptional start sites will be cloned and analyzed. From our previous studies, osteoclasts detected by TRAP staining are located along the regenerated fin rays after fin amputation in zebrafish. To investigate the role of ACP5 in bone resorption and regeneration, I used zebrafish as a model animal and performed transgenic assays to analyze the enhancer activities of acp5a with the aids of Tol2 transposon system and fluorescent proteins (DsRed, GFP). I have prepared several enhancer-promoter constructs with reporter genes. The plasmids were microinjected in the zebrafish eggs at one-cell stage. Eye-specific enhancer-promoter fragments were conjugated in the constructs to facilitate the observation and screening of transgenic zebrafish. In transient experiments (F0), the construct harboring fragments of upstream region (Exon1a + Exon1b) and TATA-276bp (Exon1c + Exon2) drives the reporter gene expression in the eyes and around neural and hemal arches of vertebrates, when observed at 7-10 dpf (day-post-fertilization). However, specific expression patterns of the reporter gene were not observed by the constructs with the fragments of Exon1c+Exon2 or Exon1a+Exon1b. Furthermore, stable lines (F1) were established by screening with the aid of reporter gene expression in the eyes. Three transgenic stable lines (No.2, No.7-1 and No.7-2) were obtained with the enhancer-promoter construct of the (Exon1a + Exon1b) and TATA-276bp (Exon1c + Exon2) combination. The expression patterns of the reporter gene in the stable lines (No.7-1. No.7-2) are approximately about the same as the currently known TRAP staining results, detected around the vertebrates, maxilla , mandible and fin rays at 14 dpf. In addition, fluorescent-labelled cells can be found scattered on the caudal fin rays and the scales. Moreover, during bone regeneration, fluorescent-labelled cells were also observed along the regenerated caudal fin rays, which was similar to the results of TRAP staining of our previous studies in the laboratory.