鹼性磷酸酶對斑馬魚胚胎造血作用之影響

Effects of Alkaline phosphatases on Zebrafish Embryonic Hematopoiesis

陳文銓(Wen-Chan Chen)

指導教授 ： 謝錦源 ； 許立松

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00061

摘要

近來之研究中顯示鹼性磷酸酶 (AP)在人類中有胎盤型、生殖型、腸道型、非組織專一性等形式，其中非組織專一性鹼性磷酸酶被認為主要參與骨骼之礦化及鈣化作用。然而藉由研究發現，鹼性磷酸酶除有協助骨骼的礦化及鈣化角色，在非組織專一性鹼性磷酸酶缺失的小鼠亦造成維生素的代謝失調，並大量降低腦部抑制性神經傳導物質伽傌丁氨基酪酸的濃度。鹼性磷酸酶在血腦障壁中亦可作為腦部微血管的分化指標。目前斑馬魚中已知有非組織專一性鹼性磷酸酶的存在，且在胚胎早期即有此基因之表現，因此本研究利用斑馬魚為模式動物，探討非組織專一性鹼性磷酸酶在胚胎發育過程中所扮演的角色。本研究以RT-PCR及全覆式定位雜合法(whole mount in situ hybridization)分析斑馬魚在組織的分佈，並以顯微注射方法，將斑馬魚非組織專一性鹼性磷酸酶之siRNA 與morpholino（MO）片段注射到斑馬魚未分化的單顆細胞受精卵內，結果顯示，經注射MO、siRNA片段的斑馬魚胚胎呈現發育延遲，且於幼魚時期可觀察到脊椎骨彎曲。在48 hpf利用o-dianisidine染色，則可觀察到紅血球的生成受到抑制。未來，將會用siRNAs與MO 分析非組織專一性鹼性磷酸酶在胚胎早期影響紅血球發育生成的角色。未來，將會利用多種RNA探針進一步分析在胚胎造血的作用中，鹼性磷酸酶所參與的機制。

關鍵字

鹼性磷酸酶造血作用

並列摘要

Alkaline phosphatase (AP), in the past study has been known three out of four ALP isozymes are tissue-specific - placental (PLAP), germ cell (GCAP), the third is intestinal (IAP), and the last is tissue non-specific (TNAP). The TNAP was involved into mineralization and calcification in skeleton. However, recent research showed that mice lacking TNAP die from seizures due to defective metabolism of vitamin B6, and reduced level of inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the brain. ALP has been established as a marker enzyme for a differentiated blood/brain barrier (BBB) phenotype in vivo and in vitro. It is known that TNAP existed in zebrafish, and expressed at early developmental stages of zebrafish embryos. Thus zebrafish as animal was selected as model to study the role of TNAP in zebrafish embryo development process. In this study, we analyze spatiotemporal expression of zebrafish TNAP by RT-PCR and whole-mount in situ hybridization. And the siRNA or morpholino of zebrafish TNAP were microinjected into zebrafish one-cell embryos to observe the roles of TNAP on embryogenesis. It was found that after TNAP knockdown embryos were delayed in embryogenesis, bended of notochord in larva period and decreased hematopoiesis (by o-dianisidnie staining) in 48hpf. In the future, we will use specific RNA probe to analyze the role of TNAP in hematopoiesis at zebrafish early development stage.

並列關鍵字

alkaline phosphatase ； hematopoiesis

參考文獻

7. Doty, S. B., and Schofield, B. H. (1976) Prog Histochem Cytochem 8(1), 1-38

10. Goldstein, D. J., Blasco, L., and Harris, H. (1980) Proc Natl Acad Sci U S A 77(7), 4226-4228

14. Harris, H. (1990) Clin Chim Acta 186(2), 133-150

23. Cusworth, D. C. (1958) Biochem J 68(2), 262-264

24. Harris, H., and Robson, E. B. (1959) Ann Hum Genet 23, 421-441