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    Please use this identifier to cite or link to this item: https://tkuir.lib.tku.edu.tw/dspace/handle/987654321/54075

    Title: 斑馬魚酪胺酸磷酸水解酶 (ptp4a1 & ptp4a2) 在胚胎早期的功能分析
    Other Titles: Functional analysis of zebrafish ptp4a1 & ptp4a2 during early embryonic development
    Authors: 李訓慈;Lee, Hsun-Tzu
    Contributors: 淡江大學化學學系碩士班
    Keywords: 斑馬魚;ptp4a1;ptp4a2;Zebrafish;ptp4a1;ptp4a2
    Date: 2011
    Issue Date: 2011-06-16 21:57:42 (UTC+8)
    Abstract: 第四類酪胺酸磷酸水解酶 (protein tyrosine phosphatase 4A, ptp4a) 可影響細胞的生長、分裂、移動及分化,且多表現於發育中的組織,因在腫瘤組織中表現量高,近年來研究當作癌症的生物標誌物。無脊椎動物中僅以一種形態存在 (PRL-1),演化為脊椎動物後分為三型 (ptp4a1、ptp4a2和ptp4a3)。三型 ptp4a 間的胺基酸序列相似度很高,本論文研究的脊椎動物斑馬魚,其中 ptp4a1和ptp4a2 有 81% 相似。以原位雜交法觀察斑馬魚胚胎時期 mRNA 的表現,ptp4a1 於12小時至三天的表現量最高,多表現於腦部 (包括終腦、間腦、中腦、小腦和後腦)、眼睛 (包括視網膜色素上皮層、外叢層、內叢層和神經纖維層)、耳包、鰭等部分。而 ptp4a2 則是在胚囊期至三天的表現量均高,大部份表現於心肌、肌節、肝臟、腸道、原腎管、氣動管、鰾、鰭、下顎及眼睛的水晶體外圈等位置。接著利用顯微注射反股寡核苷酸 (morpholino) 的方式,抑制內生性 ptp4a1 和 ptp4a2 轉譯。個別抑制 ptp4a1 和 ptp4a2 可觀察到基因缺陷後造成圍心腔腫大及細胞堆積的情形,同時抑制 ptp4a1 和 ptp4a2 外觀上的缺陷更加顯著。以F59 標記抗體及 cmlc2 探針觀察心臟肌肉,可知基因缺失會造成心臟肌肉形狀改變。從基因缺失會造成細胞增生訊號下降,但沒有細胞凋亡訊號產生等結果推測,ptp4a1 和 ptp4a2 缺失會造成細胞增生受阻,以至於心臟肌肉無法正常發育,心肌折疊不完全,產生心肌畸形。此外,ptp4a1 和 ptp4a2 缺失還會導致斑馬魚感覺器官,側線神經節發育異常,無法如同野生種發育出完整的側線神經節。由上述的實驗結果可知,ptp4a1 和 ptp4a2 在脊椎動物斑馬魚身上表現位置不同, ptp4a1 多表現於腦及眼睛,ptp4a2 表現於肌肉和心臟、肝臟、腸道等多種內臟組織中。胚胎時期若 ptp4a1 和 ptp4a2 缺陷,會影響到細胞增生以至於心臟肌肉發育不完全,且影響細胞移動能力,導致側腺神經節發育異常。由此可知,ptp4a1 和 ptp4a2 對斑馬魚胚胎的發育,是重要的環節之一。
    Protein tyrosine phosphatase 4A (ptp4a) family not only affect cell growth, division, migration and differentiation but serve as a “biomarker” of cancer in recent studies. Instead of only one type (PRL-1) in vertebrates, ptp4a family contains three members (ptp4a1, ptp4a2, and ptp4a3) with high sequence similarities. In this study, we focused on zebrafish ptp4a1 and ptp4a2 genes sharing 81% similarity. We performed in situ hybridization to observe the expression of ptp4a1 and ptp4a2 during zebrafish embryonic development. ptp4a1 was abundant from 12-hpf to 3-dpf, and mainly found in brain (tectum, tegmentum, telencephalon, diencephalon, mesencephalon, cerebellum, and metencephalon), eyes (retinal pigment epithelium, iris, outer plexiform layer, inner plexiform layer and nerve fiber layer), otic vesicle, and fin. On the other hand, ptp4a2 was abundant from 1-cell stage to 3-dpf, and was mainly distributed in myocardium, liver, fin, gut, myotomes, pronephric duct, pneumatic duct, swim bladder, arch and the outer region of the lens. Upon injection of antisense morpholino (ptp4a1-MO or ptp4a2-MO), pericardial edema and cell accumulation were observed in ptp4a1 or ptp4a2 knockdown embryos. Furthermore, co-injection of ptp4a1-MO and ptp4a2-MO resulted in even more significant defect. Heart malformation was revealed in ptp4a1/2 double knockdown embryos, based on immunostaining with muscle-specific F59 antibody and in situ hybridization with heart-specific cmlc2 probe. On the other hand, ptp4a1/2 double knockdown would result in disrupted neuromast development. In addition, such knockdown would decrease cell proliferation, but not lead to apoptosis. In summary, ptp4a1 and ptp4a2 showed differential expression patterns in zebrafish embryos: ptp4a1 was mainly found in brain and eyes, whereas ptp4a1 was mainly observed in muscle, heart, liver, gut and internal organs. Our knockdown data revealed that ptp4a1 and ptp4a2 affected cell proliferation and migration, leading to defects in zebrafish heart and neuromast development.
    Appears in Collections:[Graduate Institute & Department of Chemistry] Thesis

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