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研究生:蔡青宴
研究生(外文):Ching-Yen Tsai
論文名稱:小鼠Hurp基因在子宮生理與胚胎著床時所扮演角色之研究
論文名稱(外文):Mouse genetic study of Hurp : role of Hurp in uterine physiology and embryo implantation
指導教授:蔡亭芬
指導教授(外文):Ting-Fen Tsai
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:122
中文關鍵詞:母鼠不孕子宮生理胚胎著床子宮內膜基底細胞細胞增生肝癌上升蛋白
外文關鍵詞:female infertility in miceUterine physiologyembryo implantationendometrial stroma cellcell proliferationHurp
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HURP (Hepatoma up-regulated protein) 為一個與細胞週期息息相關的蛋白, 主要表現在細胞週期的G2/M 時期。HURP在人類的肝細胞癌、移形上皮細胞癌及軟組織瘤等癌症中,均發現有異常的表現。最近在細胞學的實驗研究報告中指出:HURP在細胞週期進行至G2/M時與微管構造的形成、穩定、染色體在赤道板上的排列及染色體的對稱分離相關;然而,目前對於HURP在活體內的確實生理功能或致病機轉,仍屬未知。目前,我們已經成功地產製了HURP基因剔除小鼠,藉以瞭解HURP的正常發育之生理功能及其致病的病理角色。經由HURP基因剔除小鼠的交配結果初步發現,HURP基因剔除小鼠至成鼠時並無明顯生理上的異常,這可能顯示HURP在正常小鼠發育過程中,並非絕對必要的。然而,在小鼠交配的實驗結果發現,HURP基因剔除的母鼠有完全不孕的現象;反之,公鼠的生殖能力則是正常。組織切片分析顯示HURP基因剔除的母鼠的卵巢不無異常且HRUP蛋白與mRNA的表現受到雌性激素的調節,主要表現在子宮內膜的基底細胞中;受孕的HURP基因剔除的母鼠中,其著床前胚胎的數量及其發育也無異常。我們的實驗結果顯示HURP基因剔除的母鼠子宮在受孕後無法使胚胎著床且無脫膜反應(decidual reaction, decidualization),導因於HURP基因剔除母鼠子宮內膜基底細胞(endometrial stroma cell)的細胞分裂停滯在G2-M期而造成基底細胞無法順利分裂與分化。本研究中針對HURP基因在活體中的功能進行分析,並意外地發現HURP基因在雌性子宮生理上的重要性,這樣的發現或許可以對人類的女性不孕,提供另一個研究的方向。
Hepatoma up-regulated protein, HURP, first revealed in cancer studies, is a cell-cycle associated gene with elevated expression in the G2/M phase. Cell culture studies have revealed that HURP is an essential factor required for spindle formation and chromosome congression during mitosis. However, the function of HURP in an in vivo context has not been explored. We generated a Hurp knockout (Hurp–/–) mouse to investigate the role of HURP in development under normal physiological conditions. Hurp–/– mice develop normally and are indistinguishable from their wild-type littermates. Interestingly, breeding experiments revealed that Hurp–/– females are completely infertile while the males reproduce normally. Ovulation, fertilization and pre-implantation embryo development are normal; however, the Hurp–/– females are unable to form implantation sites due to an inability to undergo the decidual reaction. This phenotype is caused by a defect in endometrial stroma proliferation that leads to implantation failure. Additionally, Hurp expression in the uterus coincides with the implantation stage and can be induced by estrogen treatment. Our results demonstrate for the first time that HURP affects endometrial stroma proliferation during implantation but is dispensable during normal development in mice; specifically HURP has an essential function in uterine biology.
Index

中文摘要…………………………………………………………… I
Abstract…………………………………………………………… II
A. Introduction 1
B. Specific Aims 10
Chapter I. Study the roles of Hurp in Uterine Physiology
C-1. Material and Methods 11
a. Generation of HURP Knockout Mouse 11
b. RNA Analysis 13
c. Generation of Rabbit Anti-Mouse Hurp Antibody 14
d. Mating, Embryo Collection and Implantation Sites 15
e. Partial Hepatectomy (PH) and Liver Regeneration 15
f. Induction of Artificial Decidualization 16
g. Estrogen treatment 16
h. Western Blot Analyses 16
i. Immunohistochemistry (IHC) 18
j. DNA Synthesis and Mitotic Index 18
k. Statistics 19
D-1. Results 20
a. Spatial and temporal expression profile of Hurp. 20
b. Generation of Hurp knockout mice. 21
c. Phenotypes of Hurp knockout mice. 21
d. Analysis of female infertility in the Hurp knockout mice 22
E-1. Discussion 29
a. The roles of HURP during development and pathogenesis 29
b. Normal development without overt phenotype in the Hurp–/– mice 30
c. The roles of HURP in uterus physiology 31
Chapter II. Study of Hurp in liver biology
C-2. Materials and Methods 35
a. Generation of Hurp Knockout Mouse 35
b. Partial Hepatectomy (PH) and Liver Regeneration 37
c. Detection of serum alanine aminotransferase (ALT) level 37
d. Immunohistochemistry (IHC) 38
e. DNA Synthesis and Mitotic Index 38
f. Statistics 38
D-2. Results 39
a. Expression of HURP in regenerating liver. 39
b. Generation of Hurp knockout mice. 39
c. Phenotypes of Hurp knockout mice. 39
d. Serum ALT level after liver regeneration 40
e. The proliferating hepatocytes in the regenerating liver. 41
E-2. Discussion 42
a. General characterization of Hurp KO-2 mice. 42
b. The roles of HURP regenerating liver. 42
F. References 44
G. Tables. 53
H. Figures……………………………………………………………58
Appendix …………………………………………………………….86
1. Journal of Biological Chemistry (2008). Hurp Deficiency Leads to Female Infertility Caused by an Implantation Defects.
2. Second anuscript…………………………………………………………..90
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