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    Please use this identifier to cite or link to this item: http://tkuir.lib.tku.edu.tw:8080/dspace/handle/987654321/51803


    Title: 第一部分 : 斑馬魚VHL基因在腎臟發育時的生物活性;第二部分 : 斑馬魚ATP2A家族在發育時的基因表現
    Other Titles: Part i : the biological fuction of Zebrafish VHL during kidney development;Part ii : development expression of Zebrafish ATP2A family
    Authors: 賴彥佑;Lai, Yen-yu
    Contributors: 淡江大學生命科學研究所碩士班
    陳曜鴻;Chen, Yau-hung
    Keywords: 斑馬魚;VHL;morpholino;腎臟發育;血管;Zebrafish;VHL;morpjolino;kidney development;vascular
    Date: 2010
    Issue Date: 2010-09-23 16:09:42 (UTC+8)
    Abstract: 論文提要內容:
    第一部分:
    VHL基因為罕見遺傳疾病馮希爾.林島氏症候群(von Hippel-Lindau syndrome)之致病基因,當其發生基因突變或缺失時將導致VHL disease的發生。VHL蛋白主要作用為藉由氧氣調控,降解已和氧氣結合之缺氧誘導因子(Hypoxia-Inducible Factor)1α、2α,故可因此調節缺氧誘導相關的種種作用機制。VHL蛋白又可與轉錄延伸因子 (Elongin)B-C、CUL2 蛋白組成VBC複合物,通過抑制RNA聚合酶II的活性直接抑制基因轉錄。VHL蛋白具有175個胺基酸,其第10至第164的胺基酸為具tumor suppress功能的domain。為瞭解斑馬魚VHL與其他脊椎動物之親緣關係,於是利用BlastN、Clustal W演算法與其他物種進行蛋白質序列比對,結果得到斑馬魚與熱帶爪蟾、雞、人類、狗、大鼠與小鼠等各物種的相似程度分別為 43%、40%、45%、45%、43%與45%。親緣關係上人類、犬科與小鼠的關係較為接近;而斑馬魚和雞、熱帶爪蟾較為接近。
      VHL disease的患者約76%會有腎臟損傷、囊腫的發生,而主要的機制尚未明朗,為了深入研究VHL如何對腎臟發育造成影響,我們選用斑馬魚作為材料,首先,我們利用野生種(wild type)、Tg(WTIb:EGFP)及Tg(gata-1:RFP)的斑馬魚以進行顯微注射VHL morpholino antisense oligonucleotide(VHL_MO)的方式阻斷VHL的轉譯,進而使內生性VHL蛋白的量下降,配合α6F抗體染色與螢光顯微鏡進行觀察,可觀察得到腎絲球有變形、囊腫等情形發生,腎小管角度發生異常且顯得較鬆散,也常有腎臟發育延遲之情形發生。故可證實當VHL蛋白的量下降時,會對於腎臟造成傷害甚至囊腫。接著,蒐集未接受及已接受VHL-MO注射之各時期的斑馬魚胚胎,進行Fli-1的原位雜交實驗。Fli-1主要表現於血管內皮細胞,可用於觀察血管新生的情形。實驗結果發現,當胚胎接受VHL-MO注射後,不論各個時期其Fli-1的表現均明顯高於未注射VHL-MO之胚胎(WT)。爲更加明確了解訊號表現的差異程度與內部情形,接著將搭配ZO-1、DAPI染色法、鹼性磷酸酶染色法(alkaline phosphatase stain)以及冷凍組織切片處理技術。觀察結果發現,接受VHL_MO注射後之胚胎內部血管新生確實更強烈,且有血管過度生長亂長的情形,腎贓細胞也很完整而無破裂等情形發生。這些實驗結果顯示接受VHL_MO注射後的胚胎,其血管新生更旺盛,發生血管新生的區域也更多,可能因此促使腎臟不正常發育而造成囊腫或變形。
    第二部分:
      ATP2A family在生物體內的主要功能為產生各種SERCA Ca2+-ATPases用以水解ATP並調控Ca2+的流動。ATP2A1會產生一種SERCA Ca2+-ATPases,可水解ATP並調控Ca2+由胞質流向肌肉漿質網內並刺激肌肉的收縮。ATP2A2a會產生另一種SERCA Ca Ca2+-ATPases,主要可刺激心肌收縮並調節心肌將Ca2+的泵出泵入。
      為瞭解斑馬魚ATP2A family與其他脊椎動物之親緣關係,於是利用BlastN、Clustal W演算法與其他物種進行蛋白質序列比對。.在ATP2A1的方面,斑馬魚之ATP2A1胺基酸序列與非洲爪蟾、雞、人類、狗、大鼠與小鼠等各物種的相似程度分別為87%、88%、87%、87%、87%與87%。親緣關係上人類、犬科與小鼠的關係較為接近;而斑馬魚和雞、非洲爪蟾較為接近。在ATP2A2a的方面,斑馬魚ATP2A2a之胺基酸序列與非洲爪蟾、雞、人類、狗、大鼠與小鼠等各物種的相似程度同樣分別為87%、88%、87%、87%、87%與87%。親緣關係上人類、犬科與小鼠的關係較為接近;而斑馬魚和雞、非洲爪蟾較為接近。由於兩者在各物種間皆有高達87%以上的序列相似性,顯示出ATP2A family在各個物種間具有相當高度的重要性與保守性。
      接著,為了瞭解ATP2A family在斑馬魚mRNA層次上的表現,於是利用斑馬魚的ATP2A1、ATP2A2a mRNA探針進行胚胎原位雜交實驗(whole-mount in situ hybridyzation)實驗,藉此以觀察ATP2A family之時空分佈,並偵測各時期斑馬魚胚胎中ATP2A family mRNA的表現。在ATP2A1方面,於1cell就有看到訊號出現,6小時時期形成胚盾(shield)時訊號充滿胚盾,12小時時期訊號充滿魚體全身,18小時時期訊號表現在18-somite上;於受精後24小時時期及31小時時期訊號表現在身體肌肉細胞上;48小時時期以後訊號表現在眼睛附近部分肌肉、下顎肌肉與身體軀幹肌肉細胞上;經由冷凍切片觀察,第3、4天可明顯看到除臉部、下顎與軀幹肌肉細胞外,在腸道內也有訊號的表現;受精5天至7天訊號表現則開始逐漸下降。
      在ATP2A2a方面,一樣於1cell就有看到訊號出現;受精後6小時、12小時與18小時時期魚體兩側接近卵黃的中胚層區域有訊號產生,且18小時時期在心臟前趨細胞(cardiac precursors )有明顯之訊號;在受精後24、31及48小時時期訊號表現在心臟與身體軀幹的肌肉細胞上,48小時時期下顎肌肉亦有訊號表現;在受精後3天至6天時期訊號表現在眼睛周圍、心臟和下顎部分肌肉上,身體肌肉上的訊號開始少了很多,只剩靠近頭部的部分訊號比較強烈。
    Abstract:
    PART i :
    von Hippel-Lindau Disease is a rare condition associated with familial cancers. It is due to a mutation of the VHL tumour suppressor gene at chromosome site 11q13, 3p26-p25. VHL can degrade hypoxia-inducible factors (HIF1a, HIF2a) in the presence of oxygen, thus regulating a lot of hypoxia responsive genes indirectly. pVHL forms a complex with elongins B and C, Cullin2, Rbx1, thus regulating RNA polymerase II and inactivating transcription. After sequence comparison, we found that the zebrafish VHL polypeptide shares sequence identities of 43%, 40%, 45%, 45%, 43% and 45% with the reported VHL of Xenopus, chicken, human, dog, , mouse and mice, respectively. Renal cell carcinoma is the leading cause of death in patients with von Hippel-Lindau disease, with a prevalence as high as 76% reported in one autopsy series. Here, we used zebrafish as an animal model to study it''s roles during early kideny developmental stage. Using morpholino knockdown approach, Our data showed that knock VHL down led to defect of pronephric duct, pronephric tubule and glomerulus. We also find that angiogenesis becoming more strongly after morpholino knockdown. Taken together, we concluded that VHL is required for zebrafish kidney and vascular development.


    PART ii:
    ATP2A family genes encode all kinds of the SERCA Ca2+-ATPases, which are intracellular pumps located in the sarcoplasmic or endoplasmic reticula of muscle cells. Those enzymes catalyze the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen, and is involved in muscular excitation and contraction. We used zebrafish as an animal model to study their roles during early developmental stage. After sequence comparison, we found that the zebrafish ATP2A1(ATP2A2a) polypeptide shares sequence identities of 87%(87%), 88%(88%), 87%(87%), 87%(87%), 87%(87%) and 87%(87%) with the reported ATP2A1 (ATP2A2a) of Xenopus, chicken, human, dog, , mouse and mice, respectively. Whole mount in situ hybridization experiments showed that ATP2A1 was first detected at 1cell embryos, and was expressed filled the entire animal pole of 6 hours post-fertilization (hpf) and 12hpf embryos. At the later stage, ATP2A1 was expressed at 18-somite of 18hpf, and was expressed at trunk muscle cells of 24hpf and 31hpf embryos. ATP2A1 was expressed at eye muscle cells, jaw muscle cells, and trunk muscle cells after 48hpf embryos. We also found that ATP2A1 was expressed at Intestinal after 72hpf embryos. Whole mount in situ hybridization experiments also showed that ATP2A2a was first detected at 1cell embryos, and was expressed at mesoderm on both sides of the embryo of 6hpf, 12hpf and 18hpf embryos and was expressed at cardiac precursors of 18hpf embryos. At the later stage, ATP2A2a was expressed at cardiac muscle cells and trunk muscle cells of 24hpf, 31hpf and 48hpf. ATP2A1 was expressed at eye muscle cells, jaw muscle cells, cardiac muscle cells and trunk muscle cells after 72hpf, but the Signal in the trunk muscle cells becomed more weak.
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