臺灣博碩士論文加值系統

先前的研究中發現Hypoxia-induced factor (hif1α) 弱化會造成GATA-binding protein 1 (gata-1) 表現下降，顯示hif1α可能調控gata-1基因的表現。相類似的情況也發生在人類血球中。gata-1的缺失會造成hematopoietic stem cells (HSCs) 的分化由gata-1所主導的erythropoiesis轉向由spleen focus forming virus proviral integration oncogene (spi1) 所主導的myelopoiesis，造成白血球增加，推測gata-1與spi1之間有著互相抑制的功能。因此本研究的目的想探討斑馬魚胚胎發育過程中，hif1α是否能透過調節gata-1間接影響spi1表現，進而控制erythropoiesis和myelopoiesis兩者的平衡。
在全覆式原位雜交的結果中，以monoocyte/macrophage 的探針lcp1 (lymphocyte cytosolic plastin 1)對spi1-mediated myeloid lineage 進行染色，發現弱化hif1α後會在22 hpf時期使 monocyte/macrophage的數量及分布區域都明顯增加，並在24 hpf時期觀察到在posterior ICM上，也有lcp1的表現。這些結果顯示當 hif1α被抑制時，會造成gata-1表現下降，間接使得spi1表現增加，促使common myeloid progenitor (CMP) 傾向於granulocyte/macrophage progenitor (GMP) 分化。目前已確認hif1α 弱化後，monocyte/macrophage確實有增加。未來將繼續以granulocyte探針mpo (myeloperoxidase)及erythrocyte探針hemoglobin進一步的分析，並進行基因救贖（gene rescue）的驗證，以確認上述推論的正確性。

Recent study revealed that depletion of hypoxia induced factor 1 alpha (hif1α) expression resulted in decrease of GATA binding protein 1 (gata1) transcription, suggesting that hif1α controls gata1 expression. Similar result was also found in humen erythrocyte. Blocking gata1 expression converted hematopoietic stem cell (HSCs) differentiation from gata1-mediated erythropoiesis to spi1-mediated myelopoiesis. There is an antagonistic regulation between gata1 and spi1 expression. It raised a possibility that spi1 transcription is controlled indirectly by hif1 through an antagonistic regulation between gata1 and spi1, which in turn specified the fate of HSC differentiation. Knockdown hif1α resulted in decrease of gata1 and increase of spil and lcp1 expression, suggesting that depleting hif1α converted HSC to spil-mediated myeloid-lineage differentiation. In summary, this study revealed that HIF1represses spi1 expression indirectly through the antagonist regulation between gata1 and spi1.

目錄
謝辭 i
摘要 ii
Abstract iv
目錄 v
壹、 緒論 1
一、 哺乳動物胚胎血液生成 1
二、 斑馬魚胚胎血液生成 1
三、 GATA結合蛋白1 (gata1)與spleen focus forming virus proviral integration oncogene (spi1/pu.1) 3
四、 研究動機與目的 4
貳、 實驗材料與方法 5
一、 實驗材料與儀器 5
二、 實驗藥品配製 8
三、 實驗方法 11
參、 實驗結果 30
一、 弱化hif1α會增加斑馬魚胚胎內單核球/巨噬細胞並減少紅血球減少 30
二、缺氧誘導因子 (hif) 1α弱化造成spi1表現上升 31
肆、 討論 33
伍、 圖表 35
圖一、弱化Hif1α會增加斑馬魚胚胎單核球/巨噬細胞lcp1的表現。 35
圖二、 hif1α弱化會抑制血紅素基因的表現。 36
圖三、hif1α 弱化對spi1表現的影響。 37
圖四、QRT-PCR分析hif1α弱化對spi1、lcp1與gata1表現的影響 38
陸、 參考文獻 39
柒、附錄 43
附錄一、初期血液生成之紅白血球分化路徑與調控基因 43
附錄二、QRT-PCR 基因引子序列 44

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