蝕骨細胞摘除實驗之結構體構築及其轉殖魚之篩選

Abstract

人體的骨骼是一個活動的組織，骨質不斷地在重整重塑，當骨頭受到賀爾蒙或是外部的機械壓力，骨頭會產生損傷，此時骨頭會進行修復作用，骨骼重塑的過程包含兩種現象，一種是造骨細胞的骨形成作用，另一是蝕骨細胞的骨吸收作用，兩者細胞間息息相關且互相調節，共同維持骨組織的動態平衡。而當骨組織失去平衡，就容易導致一些疾病，例如:石骨症、骨質疏鬆症。因此，了解造骨細胞與蝕骨細胞相互的作用機制就更為重要。目前已知的是當骨頭要進行重塑時，造骨細胞會分泌化學訊號促使未成熟的蝕骨細胞分化成多核成熟的蝕骨細胞並且移行到需要重塑的地方。這時成熟的蝕骨細胞會利用氫離子幫浦放出氫離子， 使蝕骨細胞與骨基質間形成酸性的環境，接著釋放溶小體內的酵素，溶解骨基質，當骨基質被分解後，造骨細胞會再過來進行骨頭的重建。而蝕骨細胞釋放的酵素包含組織蛋白酶K(cathepsin K)、基質金屬蛋白酶家族(matrix metallo- proteinases)、抗酒石磷酸酶(Tartrate-resistant acid phosphatase ,TRAP)，而這些酵素當中的抗酒 石磷酸酶(Tartrate-resistant acid phosphatase ,TRAP)普遍存在於蝕骨細胞中，因此可以作為蝕骨細胞的標記物。但是蝕骨細胞是否也會釋放出訊號促使造骨細胞重建骨頭，與其相關機制並不是很清楚，因此本實驗的目標是藉由細胞摘除的方式探討蝕骨細胞的功能。我們利用了Acp5a 和Acp5b(TRAP)啟動子，附帶綠色的螢光標記蛋白(hrGFP)，並且加上還原酶reductase (由Dr. Peter F.Searle 實驗室取得)的質體作斑馬魚轉殖實驗。先前實驗室資料得知蝕骨細胞大致分布在頭骨、鰓蓋、尾柄、背鰭、臀鰭以及脊椎骨上下緣。三天可以看到眼睛水晶體促進子標記螢光訊號蛋白(hrGFP)，一周可以看到頭部、尾柄也開始產生螢光訊號。兩周脊椎骨上下緣有開始有螢光的表現，三周後背鰭、胸鰭、臀鰭也開始有螢光訊號。本實驗先建立帶有Acp5a 和Acp5b 啟動子，附帶綠色的螢光標記(hrGFP)，並且加上還原酶(reductase)的轉殖斑馬並觀察記錄其蝕骨細胞的螢光時空分布。在Acp5b 啟動子組編號A 組在7 天可以觀察到咽弓、胸鰭基部、耳石有綠色螢光訊號，並在第10 天還是可以持續觀察到綠色螢光訊號。而在Acp5a 組在第7 天可以觀察腎臟組織有綠色的螢光訊號，並且在第10 天也可以發現螢光訊號在腎臟中段表現。 未來利用甲硝唑(Metronidazole)上的硝基被還原酶還原時會產生產生硝基咪唑(Nitrosoimidazole)並誘導細胞凋亡。在後續的實驗，將轉殖斑馬魚進行泡藥摘除實驗，把魚浸泡在Metronidazole(MTZ)中，觀察蝕骨細胞凋亡對骨細胞與骨頭型態的影響，並使用Alizarin red(染硬骨) 與alcian blue(染軟骨)染色觀察硬骨與軟骨型態。更進一步把各個蝕骨細胞表現之時間點，進行泡藥摘除實驗，並且染色觀察各階段蝕骨細胞的摘除對各個階段魚骨頭發育的影響。

Bone is an active tissue in human body. When the bones receive hormones or under external mechanical pressure, the bones will be remodeled. Body will try to repaired the bone simultaneously. The procedures of bone remodeling involve bone destrucetion,absorption, and bone regeneration. Two different types of cells, osteoblasts and osteoclasts, participate in bone remodeling. During remodeling, osteoblasts will release some signal factors to recruit pre-osteocalst to the bone surface where bones need to be rebuilt, and then pre-ostoeclast will differentiate into mature multinucleated osteocalsts and adsorb the bones. At the same time, mature osteoclasts use hydrogen ions pump to release hydrogen ions, which forms an acidic environment between the osteoclasts and the bone matrix, and then release the enzymes to dissolve the bone matrix. When the bone matrix is decomposed, osteoblast will be recruited and rebuild the bones.The enzymes released by osteoclasts include matrix metalloproteinases, and tartarresistant acid phosphatase (TRAP), cathepsin K. Among them, the tartar-resistant phosphatase (TRAP) is generally found in osteoclasts . Therefore, TRAP can be used as an osteoclasts marker. However, osteoclasts release some signals to promote bone remodeling , the related mechanisms are elusive. The goal of this experiment is to explore the function of osteoclasts by cell ablation. I used the constructs of osteoclast-specific gene promoter Acp5a and Acp5b with a green fluorescent protein as a reporter gene labelling osteoclasts in conjugation with reductase cDNA to ablate osteoclasts (obtained by Dr. Peter F. Searle Laboratories).Then these constructs were microinjected into one-cell stage of zebrafish embryos.The expressions of hrGFP drived by Acp5a or Acp5b promoter are observed by green fluorescent protein. From our previous laboratory data showed that the osteoclasts reside in the skull,the sac, stalk, dorsal fin, anal fin, and upper and lower edges of the ventral spine. In this experiment, I build two types of transgenic zebrafish. One is Acp5a promoter-reductase-IRES-hrGFP transgenic zebrafish, the other is Acp5b gene promoter-reductase-IRES-hrGFP transgenic zebrafish. In Acp5a promoter transgenic zebrafish stable line (No.20) I observed the hrGFP-labelled osteoclasts were broadly located in the primary hematopoietic tissue and kidney at 7dpf , middle site of kidney at 10dpf. In Acp5b promoter transgenic zebrafish stable line (No.A) I observed the hrGFP-labelled osteoclasts were broadly located in the pharyngeal arch, otolith and the base of pectoral fins at 7dpf. In the future, the transgenic zebrafish will be used in reductase experiments. When Metronidazole reacts with reductase, the nitro group of Metronidazole is reduced and transform to Nitrosoimidazole, which produces toxicity and causes osteoclast apoptosis.In the future experiments, we may observe the effects of osteoclast ablation on hard bone and cartilage with Alizarin red and alcian blue staining. Moreover, we may observe the ablation of osteoclasts at each development stage.