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研究生:林世杰
研究生(外文):Lin shih-chieh
論文名稱:腦下垂體腫瘤轉型基因-1在子宮肌瘤的表現之探討
論文名稱(外文):Expression of Pituitary Tumor Transforming Gene-1(PTTG1) in Uterine Leiomyoma
指導教授:陳麗玉陳麗玉引用關係
指導教授(外文):Lih-Yuh C. Wing
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:82
中文關鍵詞:腦下垂體腫瘤轉型基因-1子宮肌瘤
外文關鍵詞:PTTG1leiomyoma
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  腦下垂體腫瘤轉型基因-1(PTTG1)最早是從大鼠腦下垂體腫瘤細胞株中分離出來。它有許多功能,除了可促使體外培養的細胞轉型,而且在鼠體內可促使腫瘤的生成;另外,腦下垂體腫瘤轉型基因-1為調控細胞分裂的調控者,也可促進纖維母細胞生長因子-2(FGF2)的分泌而纖維母細胞生長因子-2也可刺激腦下垂體腫瘤轉型基因-1的表現。子宮肌瘤是女性最常見的良性腫瘤,它是由正常的子宮肌層所演變而來的腫瘤;有關子宮肌瘤的致病機轉尚未清楚,但是許多的研究結果皆顯示子宮肌瘤的生長是受到女性賀爾蒙以及生長因子的調控。本論文的目的是要探討腦下垂體腫瘤轉型基因-1在子宮肌瘤致病機轉所扮演的角色。因此,我們取得子宮肌瘤以及配對的正常子宮肌層組織來分析探討了腦下垂體腫瘤轉型基因-1,纖維母細胞生長因子-2以及細胞增生三者之間的相互關係。實驗檢體來源主要是病人接受去子宮或者是摘除子宮肌瘤手術所取得。藉由標準曲線核糖核酸定量以及西方轉漬法分析結果顯示,子宮肌瘤的腦下垂體腫瘤轉型基因-1的核糖核酸(RNA)以及蛋白質(protein)的表現皆明顯高於正常的子宮肌層。雖然在月經週期的增生期以及黃體期,腦下垂體腫瘤轉型基因-1的核糖核酸表現皆有顯著增加,但是經由初級培養的子宮肌瘤細胞在分別處理雌激素(estrogen)以及黃體激素(progesterone)後,其腦下垂體腫瘤轉型基因-1的表現並未增加。此外,我們的實驗結果顯示,子宮肌瘤的組織比正常的子宮肌層表現更多的纖維母細胞生長因子-2以及增生性細胞核抗原(PCNA),而這兩種蛋白的表現與腦下垂體腫瘤轉型基因-1的表現皆有顯著正相關性。因此我們更進一步探討是否纖維母細胞生長因子-2可以調控腦下垂體腫瘤轉型基因-1的表現,實驗結果顯示子宮肌瘤細胞在纖維母細胞生長因子-2的處理後,腦下垂體腫瘤轉型基因-1的表現以及細胞生長皆有顯著的增加。為了更進一步探討腦下垂體腫瘤轉型基因-1在子宮肌瘤的功能為何;我們接著利用基因轉殖的方法將腦下垂體腫瘤轉型基因-1的表現載體外送至子宮肌瘤細胞中,發現除了可刺激纖維母細胞生長因子-2表現增加之外,還可促使細胞週期產生改變。總而言之,我們發現腦下垂體腫瘤轉型基因-1以及纖維母細胞生長因子-2的表現增加跟子宮肌瘤細胞增生有相關;而在子宮肌瘤細胞,腦下垂體腫瘤轉型基因-1與纖維母細胞生長因子-2兩者的表現似乎存在一個正迴饋機制,而此機制對子宮肌瘤的生長可能扮演一個重要的角色。
  Pituitary tumor transforming gene-1 (PTTG1), originally isolated from rat pituitary tumor cells, can transform cells in vitro and is tumorigenic in vivo. It also serves as a regulator of mitosis and regulates secretion of fibroblast growth factor-2 (FGF2) that in turn stimulates PTTG1 expression. Uterine leiomyoma, derived from myometrium, is the most common benign uterine tumor in women. Although the cause of leiomyoma is still unclear, substantial evidences have shown that the growth of tumor is affected by ovarian hormones and growth factors. To investigate the potential role of PTTG1 in pathogenesis of uterine leiomyoma, we sought the interrelationship among the expression of PTTG1, FGF2 and cell proliferation in uterine leiomyomas and the matched myometrial tissues. Tissues were collected from the patients for hysterectomy or myomectomy. Our results showed that the expression of mRNA and protein of PTTG1 was higher in leiomyoma than that in myometrial tissues by using standard curve-quantitative competitive RT-PCR and Western blot, respectively. Even though the expression of PTTG1 mRNA was increased at both proliferative and secrectory phase of menstrual cycle, neither estrogen nor progesterone treatment induced expression of PTTG1 mRNA in cultured leiomyomal cells. The expression of FGF2 and PCNA were elevated in leiomyoma tissues, and their expression is positively correlated with PTTG1 expression. In addition, FGF2 treatment of cultured leiomyomal cells induced PTTG1 expression and also stimulated cell cycle progression. Transient transfection of PTTG expression vector into leiomyomal cells also induced FGF2 mRNA expression. Taken together, we have shown that the elevated expression of PTTG1 and FGF2 is correlated with increased cell proliferation in
leiomyoma. There may exist a positive auto-feedback regulation of PTTG1 and FGF2 that may play important roles in the growth control of uterine leiomyoma.
中文摘要.............................................8
I. Introduction......................................12
1.1. Uterine leiomyoma............................................12
1.2. Factors affect the development of leiomyomas...........................................13
1.2.1. Ovarian steroid hormones.............................................13
1.2.2. Growth factors..............................................14
1.2.3 Genetics and the development of uterine leiomyomas...................................16
1.3. Pituitary Tumor Transforming Gene-1 (PTTG1).....16
1.3.1 Human PTTG1 Gene family........................17
1.3.2 Structure of PTTG1.............................17

1.3.3 Function of PTTG1..............................18
1.3.3.1 As transcription factor......................18
1.3.3.2 As securin...................................19
1.3.3.3 As modulator.................................20
1.3.4 Phosphorylation of PTTG1.......................21
1.3.5 Regulation of PTTG1............................21

II.Materials and Methods.............................23
Tissue collection....................................23
Cell culture.........................................23
Isolation of total RNA...............................25
RNA Gel..............................................27
Primer design........................................27
Standard-curve, quantitative and competitive RT-PCR (SC-QC-RT-PCR).....................28
Minipreparation of plasmid DNA.......................29
Construction of expression vector....................30
Transient transfection...............................31
In vitro transcription (IVT).........................32
Protein extraction and immunoblotting (IB)...........33

III. Results.........................................39
Expression of PTTG1 was elevated in leiomyoma compared with normal myometrial tissues............................39
Ovarian steroids did not stimulate PTTG1 expression in cultured leiomyomal cells......39
Expression of PCNA and FGF2 were increased in leiomyomal tissues and their expressions were correlated with PTTG1 expression..........................................40
FGF2-induced PTTG1 expression was cell cycle dependent in leiomyomal cells..........41
Transient transfection of PTTG1 into leiomyomal cells changed expression.................42
4
Transient transfection of PTTG1 into leiomyomal cells increased FGF2 and VEGF mRNA expression..........................................42

IV. Discussion......................................60

V. References.......................................64

VI. Appendix........................................70

Reagents and buffers used in cell cultu.............70
Reagents and buffers used in isolation of plasmid DNA..72
Reagents and buffers used in RNA isolation and RNA gel..73
Reagents and buffers used in Western blot...............76
Reagents and buffers used in electrophoresis............79

About the author........................................82
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