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系統識別號 U0026-1508201622190600
論文名稱(中文) 前胸腺素在多囊性卵巢症候群致病機轉中扮演的角色
論文名稱(英文) Role of Prothymosin α in the Pathogenesis of Polycystic Ovary Syndrome
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 104
學期 2
出版年 105
研究生(中文) 劉宛艷
研究生(英文) Wan-Yen Liu
學號 S16031120
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-許耿福
口試委員-歐弘毅
口試委員-李哲欣
中文關鍵字 前胸腺素  過氧化體增生受體  多囊性卵巢症候群 
英文關鍵字 Prothymosin α  peroxisome proliferator activated receptor γ  Polycystic Ovary Syndrome 
學科別分類
中文摘要 多囊性卵巢症候群是常見的生殖內分泌系統異常疾病之一,10位生育年齡的女性中就有1人會罹患此疾病。一般常見的診斷標準三項中符合至少兩項時,即可診斷為多囊性卵巢症候群。第一項為血液中雄性素睪固酮過多;第二項則是排卵數量減少或不排卵導致月經不規則,反覆流產甚至造成不孕;第三項則是在超音波檢查上可以觀察到卵巢有囊泡形成,並且會呈現 ”珍珠項鍊串”的影像。病患中一半以上有肥胖的現象,且高達70%的患者有胰島素抗性產生。前胸腺素是一種結構簡單且小的酸性核蛋白。我們實驗室之前發現它不但會降低組織蛋白去乙醯酶和組織蛋白的結合,並且增加組織蛋白乙醯化,而且在過度表現前胸腺素的小鼠身上可以看到胰島素抗性現象的發生。本研究目的為探討前胸腺素在多囊性卵巢症候群病程發展中扮演的角色。我們發現多囊性卵巢症候群病患血液中測到較高濃度的前胸腺素。我們接著建立去氫皮質酮誘導多囊性卵巢症候群的小鼠動物模式,並在小鼠動物模式上可以觀察到跟人類的多囊性卵巢症候群相同的病理特徵,包含月經週期紊亂、雄性素過高、多囊性卵巢、體重過重和胰島素抗性。同時我們在多囊性卵巢症候群小鼠的卵巢和脂肪組織中看到前胸腺素的蛋白表現高於正常小鼠。此外我們同樣給予過度表現前胸腺素的小鼠去氫皮質酮,發現會比正常小鼠產生更嚴重的多囊性卵巢症候群病徵。在過度表現前胸腺素小鼠的肺臟微陣列資料顯示過氧化體增生受體 (peroxisome proliferator activated receptor γ; PPARγ) 調控的下游基因表現量下降。因此我們假設前胸腺素是透過調控過氧化體增生受體在多囊性卵巢症候群致病機轉上扮演重要角色。實驗結果顯示前胸腺素會調控過氧化體增生受體活化程度的表現,並且會直接和過氧化體增生受體結合,增加過氧化體增生受體的乙醯化,進一步降低過氧化體增生受體的活性。由以上的實驗結果顯示,前胸腺素會抑制過氧化體增生受體的活性,而此結果可能導致給予去氫皮質酮的小鼠產生多囊性卵巢症候群。此研究不但發現了新的多囊性卵巢症候群致病機轉,並且有可能對於未來治療多囊性卵巢症候群提供一個新的治療辦法。
英文摘要 Polycystic ovary syndrome (PCOS) is one of the endocrine disorders of the female reproductive system and affects 6%-10% of women worldwide. Clinical diagnostic features of PCOS are infertility, hyperandrogenism, and polycystic ovary. In addition, 50%-70% of patients with PCOS show obese and insulin resistance. Prothymosin α (ProT), an acidic nuclear protein, plays a role in the acetylation of histone and non-histone proteins by decreasing its association with histone deacetylase. We have recently reported that overexpression of ProT contributes to the development of insulin resistance. In the clinical samples, we found that ProT levels were significantly higher in the PCOS group compared with the non-PCOS group. The aim of this study was to investigate whether ProT is involved in the pathogenesis of PCOS. Next, we established a dehydroepiandrosterone (DHEA)-induced PCOS mouse model. DHEA-treated mice exhibited many characteristics of the human condition, including disrupted estrous cycles, hyperandrogenism, polycystic ovary, overweight, and insulin resistance, as well as expressed higher levels of ProT in the ovarian and adipose tissues. In addition, ProT transgenic mice had more severe PCOS than wild-type mice after DHEA treatment. Our microarray data from the lung tissues of ProT transgenic mice revealed downregulation of genes that are involved in the peroxisome proliferator activated receptor γ (PPARγ) signaling pathway. Therefore, we hypothesized that ProT may play a pivotal role in the pathogenesis of PCOS through PPARγ signaling. We observed that ProT overexpression reduced the transactivation activity of PPARγ. ProT could interact with PPARγ and reduce PPARγ activity by enhancing its acetylation. Collectively, these results suggest that ProT may inhibit PPARγ activation, leading to PCOS in DHEA-treated mice. This study may not only shed lights on the pathogenesis of PCOS, but also provide novel therapeutic strategies for PCOS.
論文目次 Abstract………………………………………………….…………………………...I
Chinese abstract……………………………………………………………………..II
Acknowledgement…………………………………………………………………III
Introduction……………………………………………………….………………....1
Materials
1. Plasmids
1.1. Expression vectors……………………………………………….……………...5
1.2. Short hairpin RNA (shRNA)……………………………………………………5
2. Cell lines……………………………………………….………………………...6
3. Primers…………………………………………………….……………………..6
4. Antibodies
4.1. Primary antibodies……………..……………………………….………...……..7
4.2. Second antibodies…………………………………………………………...…..7
4.3. Others………………………………………………………………..…………..7
5. Compounds
5.1. Recombinant proteins……………………………..…………………..……...…8
5.2. Chemical compounds……………………………………………………………8
6. Reagents
6.1. Bacterial growth media………………………………………………...…..……9
6.2. Cell lysis buffers………………………………………………………...……..10
6.3. Immunoblotting buffers………………………..………………..……………..11
6.4. Reporter assay buffers……………………………………………………….…12
7. Bacteria………………………………………………………..………………..12
8. Mice……………………………………………………………...……………..12
Methods
1. Cell culture……………………………………….…………….……………...13
2. Transfection…...................................................................................................13
3. Lentivirus production….....................................................................................13
4. Reporter assay…………………………………………………….…………...14
5. Immunological assay………………………………………….……………....14
6. Real-time quantitative RT-PCR analysis……………………………..……..…15
7. Animal experiments
7.1. Dehydroepiandrosterone (DHEA)-induced PCOS mouse mode……………...16
7.2. Assessment of estrous cycle……………………………………………….…..17
7.3. Serum testosterone and insulin resistance index tests…………………….…...17
7.4. Immunohistochemistry………….……………………….……………….……17
7.5. Classification of ovarian follicles………………………………….…………..18
8. Genotyping……………………………………………….………………...…18
9. Statistical analysis…………………………………….……….……….......…19
Result
ProT levels are elevated and positively correlated with 2-hour post-load insulin in the women with PCOS…………………………………………………………………20
ProT transgenic mice exhibit a PCOS-like symptom………………………………20
DHEA-treated mice exhibit a PCOS-like phenotype…………………………….…21
DHEA-treated mice express elevated levels of ProT in the ovarian and adipose tissue………………………………………………………………………………..22
ProT transgenic mice have a more severe PCOS phenotype…………………….…23
ProT regulates PPARγ transactivation activity………………………………......…24
ProT can interact with PPARγ and reduce PPARγ activity by enhancing its acetylation………………………………………............................................…….24
Discussion………………………………………….………………………………26
Conclusion……………………………………………………………………...….28
References…………………………………….……………………………………29
Figure legends……………………………………………………………….……..35
Appendix……………………………………………….…………………………..46
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