臺灣博碩士論文加值系統

血管新生在胚胎發育以及一些病理情形如傷口癒合、 腫瘤生長及轉移中都扮演重要的角色。因此，了解這些生理以及病理情形中血管新生如何被調控就顯得非常重要，可藉由了解這些調控機制，發展出治療或是控制這些疾病的方法。Tie2-Cre 是在內皮細胞中專一表現 Cre DNA重組酶的基因轉殖小鼠株。 利用這一隻小鼠，Dr You已經成功的剔除小鼠內皮細胞中的COUP-TFII基因。(Nature 435, 98-104)。但是這隻突變小鼠會在胚胎時期12天前死亡，使得無法研究COUP-TFII在之後胚胎發育時期以及成體內皮細胞中的功能。為了克服這樣的限制，我們希望能藉由建立一隻Tie2-CreERT2基因轉殖小鼠株，在時間上控制COUP-TFII剔除事件的發生。Tie2-CreERT2轉殖基因中含有 Tie2 啟動子/增強子，能驅動Cre以及突變型的人類雌激素受體配體結合區(mutated human estrogen receptor LBD, ERT2)形成的融合蛋白專一地在內皮細胞中表現，並且CreERT2融合蛋白能受到Tamoxifen的誘導而進入細胞核執行其功能。經由原核胚顯微注射，我們得到7隻原源種基因轉殖小鼠(founder)，分別命名為Tg1到Tg7。為了分析Cre受到Tamoxfien誘導後的重組酶活性，我們將 7 隻原源種基因轉殖小鼠與R26R Cre 報導小鼠，以及COUP-TFIIflox/flox 小鼠配種，得到雙基因轉殖小鼠進行Cre活性的分析。在分析的5個小鼠株中，發現Tg1基因轉殖小鼠株受到Tamoxfien誘導後，能在內皮細胞中專一地展現Cre的活性。利用Tg1小鼠株剔除成鼠內皮細胞中的COUP-TFII，我們也初步分析了突變小鼠在成體血管新生中是否有缺陷。我們建立的這一隻Tie2-CreERT2基因轉殖小鼠株可作為一個很好的動物模式，用以研究COUP-TFII及其他內皮細胞專一性表現的蛋白在血管新生過程中所扮演的功能。

Angiogenesis plays a critical role during embryonic development as well as in diseases such as wound healing, retinopathy and cancer growth and metastasis. Thus, it is important to understand how angiogenesis and neovascularization are regulated in vivo, in order for therapeutic means to be developed to prevent or control the progressing of these important diseases. Tie2-Cre, the endothelial-specific transgenic mouse line, has been successfully used for conditional gene inactivation in our previous study (Nature 435, 98-104). However, the early embryonic lethality prevents us from further addressing COUP-TFII functions at later developmental stage. To overcome this limitation, the goal of this project is to generate an inducible endothelial-specific Tie2-CreERT2 transgenic mouse line. Tie2-CreERT2, harboring Cre and mutated estrogen receptor LBD (ERT2) fusion protein under the control of an endothelial-specific Tie2 promoter/enhancer, which can be induced by synthetic estrogen antagonist tamoxifen. This inducible Cre recombinase of transgenic mouse model can further facilitate conditional gene knockout analysis to assess gene function at specific time points in a highly controlled manner. Seven Tie2-CreERT2 transgenic founders were obtained after pronuclear microinjection. To characterize these mouse lines, all of the founders were crossed with R26R reporter or COUP-TFflox/flox. The inducible expression profiles of these Cre lines by proper dosage of tamoxifen were characterized. One of the five lines shows high excision event in endothelium of various adult tissues. This transgenic line is now crossed with floxed COUP-TFII mice and tamoxifen are administrated to generate endothelial-specific null mice at adult stage. The angiogenesis during wound healing and cancer growth are now been investigating. This mouse line will be a valuable mouse model for exploring the biology and pathogenesis of COUP-TFII and endothelial specific genes during vascular formation.

目錄
中文摘要..................................................1
英文摘要..................................................2
壹、緒論
1-1 COUP-TFs 家族成員.....................................4
1-2 Cre/loxP 基因剔除系統.................................6
1-3 COUP-TFII於胚胎內皮細胞中之功能.......................8
1-4 成體血管新生.......................................................10
1-4-1 腫瘤血管新生.......................................12
1-4-2傷口癒合............................................13
貳、實驗材料與方法
2-1實驗材料..............................................16
2-1-1藥品與器材..........................................16
2-1-2 本實驗使用之質體與細胞株...........................17
2-1-3 本驗使用之小鼠品系.................................17
2-1-3-1 COUP-TFII 之條件式基因修飾小鼠(COUP-TFIIflox/flox)
及COUP-TFII/LacZ標的基因轉殖(knock-in)小鼠.......17
2-1-3-2 Rosa26 Cre reporter(R26R) 報導基因小鼠...........18
2-2 方法.................................................19
2-2-1 Tie2-CreERT2質體構築...............................19
2-2-2 Tie2-CreERT2 基因轉殖小鼠.......................................................19
2-2-3 細胞轉染...........................................20
2-2-4 於HeLa細胞中以4OHT誘導CreERT2活性..................20
2-2-5 產製 Tie2-CreERT2/+;COUP-TFIIflox/+ 及Tie2-
CreERT2/+;R26RTg/+小鼠.............................20
2-2-6 聚合酶連鎖反應確認小鼠基因型.......................21
2-2-7 4-Hydroxytamoxifen誘導Cre重組酶之酵素活性........24
2-2-8 Tamoxifen誘導Cre重組酶之酵素活性..................24
2-2-9 小鼠全組織器官 X-Gal 染色..........................25
2-2-10 小鼠組織器官之冷凍包埋、組織切片及X-Gal 染色......25
2-2-11 石蠟包埋與組織切片................................26
2-2-12 Lewis 肺腫瘤細胞之培養............................27
2-2-13 以Lewis 肺腫瘤細胞誘發小鼠腫瘤形成................27
2-2-14 傷口癒合實驗......................................28
2-2-15 免疫組織化學染色..................................28
參、結果
3-1 分析COUP-TFII 在成鼠內皮細胞中表現情形...............30
3-1-1 以 COUP-TFII/LacZ 標的轉殖基因小鼠分析COUP-TFII在
成鼠內皮細胞中表現情形.............................30
3-2 建立並分析 Tie2-CreERT2 基因轉殖小鼠.................31
3-2-1 構築 Tie2-CreERT2 表現質體.........................31
3-2-2 在HEK293T細胞中測試 Tie2-CreERT2 表現質體受 4OHT
誘導情形...........................................31
3-2-3 建立Tie2-CreERT2 基因轉質小鼠株...................32
3-2-4 以4OHT誘導CreERT2活性之分析........................32
3-2-5 以Tamoxifen誘導CreERT2活性之分析...................33
3-2-6 利用Rosa26 Cre reporter 基因轉殖小鼠分析Tamoxifen
對於CreERT2活性之誘導..............................34
3-3-1 COUP-TFII在成體新生血管中的表現....................35
3-3-2 內皮細胞專一性剔除COUP-TFII對於腫瘤生長大小之分析..36
3-3-3 內皮細胞專一性剔除COUP-TFII對於懷孕母鼠胎盤形成影
響之分析...........................................37
3-3-4 內皮細胞專一性剔除COUP-TFII對於傷口癒合中血管新生
之影響.............................................38
肆、討論
4-1 在HEK293T細胞中測試 Tie2-CreERT2 表現質體受 4OHT
誘導情形.............................................39
4-2 4OHT以及Tamoxifen誘導CreERT2活性之分析...............39
4-3 COUP-TFII在成體新生血管中的表現.....................42
4-4 內皮細胞專一性剔除COUP-TFII對於腫瘤生長大小之分析 ...43
4-5 內皮細胞專一性剔除COUP-TFII對於懷孕母鼠胎盤形成影響
之分析...............................................43
4-6 內皮細胞專一性剔除COUP-TFII對於傷口癒合中血管新生之
影響.................................................44
伍、參考文獻.............................................45
陸、附圖.................................................49

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