臺灣博碩士論文加值系統

Pdia4蛋白隸屬於蛋白質雙硫異構酶 (Protein disulfide isomerase； PDI)家族，在細胞內的內質網及細胞膜均有表現。CGHC活性催化位是蛋白質雙硫異構酶家族的主要活性結構，而其功能主要在於幫助蛋白質雙硫鍵的鍵結、幫助蛋白質正確的摺疊形成穩定的結構。近年來，許多研究也陸續提出了其他非內質網所提供的功能，如：影響生長與死亡、血液凝集、轉移、免疫功能、受精作用…等。但Pdia4的生理功能為何仍有待釐清。
為此，本論文著手進行Pdia4在細胞內的分部觀察及Pdia4基因剔除小鼠的製造。以西方墨點法及共軛焦顯微鏡的觀察下，Pdia4基因普遍存在多數細胞中，且細胞核、質、膜皆有分布，以細胞質、膜為主。觀察Pdia4基因表現量時發現其在癌細胞有較高的表現量，推測可能與癌細胞的特性相關。
為了進一步了解釐清Pdia4基因在活體內的功能與重要性，利用Pdia4基因剔除小鼠做為模式動物。由觀察Pdia4的基因剔除小鼠顯示小鼠似乎正常，但在雄性子代比例偏低。這部分基因的缺陷可能會影響性別決定或受精作用。

Pdia4 belongs to the protein disulfide isomerase (PDI) family. Several lines of evidence showed that this protein distribution in the cell membrane and endoplasmic reticulum. This PDI family contains one to three CGHC motifs for the catalysis of disulfide bond formation and protein folding in ER. It was proposed that the PDI family, there are has non-ER functions, including growth or death, immunity function and fertilization. However, little is known about the function of Pdia4.
In this study, we first examined the cellular distribute and expression level of Pdia4. Western blot and confocal microscopy showed that Pdia4 was expressed in membrane, cytoplasm and nucleus. We also found that Pdia4 expression was up-regulation in leukemia cell lines as well as other tumor cells. To further investigate the in vivo role of Pdia4, we generated Pdia4 knockout mice. We found that Pdia4 knockout mice appeared healthy mice. However, a low rate of male in Pdia4 knockout mice was observed. This suggest that Pdia4 is involved in sex determination or fertility.

目 錄

謝 辭 ....................................................i
中文摘要 ................................................iii
英文摘要 ................................................ iv
目 錄 ....................................................v
前 言 ....................................................1
蛋白質雙硫異構酶 .........................................1
基因轉殖、剔除鼠發展應用 ..................................4
材料與方法 ................................................8
第一部份、Pdia4蛋白在T淋巴球細胞分布及表現分析 ................8
一、 細胞培養 ...........................................8
二、 初代T淋巴球細胞的收集 ...............................9
三、 細胞蛋白質萃取 ....................................10
四、 蛋白質電泳分析與西方墨點法 ..........................11
五、 T淋巴球細胞免疫螢光染色 ............................12
第二部份、Pdia4 基因剔除鼠建立 ..............................13
一、 Pdia4基因剔除重組質體之構築 ........................13
二、 細菌人工染色體DNA大量製備 ..........................14
三、 “基因標的 (gene targeting)”實驗...................15
四、 聚合酶鏈鎖反應 (PCR) ..............................16
五、 胚胎幹細胞篩選 ....................................16
六、 Pdia4 嵌合鼠的製造及育種 ...........................16
七、 基因剔除小鼠尾巴組織蛋白質萃取 ......................17
八、 基因剔除小鼠骨髓、胸腺、脾臟細胞製備..................17
九、 流式細胞儀分析基因剔除小鼠淋巴球細胞族群分布...........18
結 果 ...................................................20
一、 觀察Pdia4蛋白在不同型細胞內部的表現情形 ..............20
二、 觀察Pdia4蛋白在不同型T淋巴球細胞內部的表現情形 ........21
三、 觀察Pdia4蛋白在T淋巴球細胞內部的分布情形 .............21
四、 製造條件式Pdia4基因剔除小鼠胚胎幹細胞株 ..............22
五、 條件式Pdia4基因剔除嵌合鼠的製造與繁殖 ................23
六、 Pdia4基因剔除鼠的製造與繁殖 ........................23
七、 Pdia4基因剔除鼠其Pdia4蛋白表現量分析 ................25
八、 分析Pdia4基因剔除鼠血球細胞族群數量 .................25
討 論 ....................................................27
一、 Pdia4蛋白在不同型的腫瘤細胞相較正常細胞具有高度的表現...27
二、 Pdia4蛋白在T淋巴球細胞內部細胞核、細胞質、細胞膜皆分布..30
三、 傳統式Pdia4基因剔除小鼠子代活存率下降、性別發育不均....31
四、 總結..............................................34
圖表、附錄 ................................................36
參考文獻 .................................................57

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