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研究生:黃金緯
研究生(外文):Chin-Wei Huang
論文名稱:雄性素受體對於肌肉前驅細胞決定的功用
論文名稱(外文):The function of androgen receptor on the myogenic determination of pluripotent progenitor cells
指導教授:陳盛良
指導教授(外文):Shen-Liang Chen
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文出版年:2005
畢業學年度:94
語文別:英文
論文頁數:96
中文關鍵詞:雄性素受體肌肉前驅細胞
外文關鍵詞:androgen receptormyogenic determination
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雄性素的補充能增加肌肉的發達是眾所皆知的事;舉例說明,像是性腺能力低的年輕人或老人攝取雄性素後都能促進肌肉蛋白的生成,少數職業運動員更是藉由雄性素增強肌肉能力來達到更好的成績;雖然生理現象是很明顯的, 但是在分子生物的機制上 ,雄性素是如何調控前趨細胞進而促使其走向肌肉細胞仍然是個未知 。 MRFs (Myogenic regulatory factors)家族包括MyoD, Myf-5, myogenin和MRF4等轉錄因子,它們都是調控肌肉細胞分化的肌肉專一性(muscle-specific) 轉錄因子。目前已經有報告指出在分子機制上Wnt3a會透過β-catenin進而去誘導Mox1、 Pax3、 Gli2 的表現,這些因子再進而調控MRFs的表現,我們藉由此項報告推論雄性素受體可能會在Wnt3a下游透過中間的任何因子進而去調控MRFs家族參與肌肉分化;首先為了使雄性素受體對於肌肉分化的影響更明顯,我們使用了兩種方法在細胞中大量表現雄性素受體,第一種是adenovirus,第二種是retrovirus。C3H 10T1/2細胞株分別被帶有雄性素受體cDNA的adenovirus 或retrovirus 感染,被感染的C3H 10T1/2細胞株則能分別暫時性或永久性的表現雄性素受體。5-azacytidine處理過的C3H 10T1/2細胞株已經被前人實驗證實能夠被誘導走向肌肉細胞路線,因此我們用5-azacytidine處理會大量表現雄性素受體的C3H 10T1/2細胞株,在分別有無處理雄性素情況下探討解析雄性素受體是如何參與肌肉分化的路徑。非常有趣地,我們發現 5-azacytidine處理過大量表現雄性素受體的C3H 10T1/2細胞株在沒有雄性素的處理下能夠走肌肉路線的比例比有雄性素處理下更高。此外,flutamide,一種anti-androgen,竟然無法抑制大量表現雄性素受體的C3H 10T1/2細胞株走向肌肉路線;相反地,反而更加促使C3H10T1/2細胞株走向肌肉體系(myogenic lineage)。因此,我們推測雄性素受體是在細胞質中作用促進前趨細胞走向肌肉細胞,並且可能利用Wnt3a-β-catenin的路徑來調控肌肉體系的走向,我們也推測AR可能在細胞質中透過影響signalling pathway來調控MRFs。
It is well known that androgen supplementation can increase muscle strength. Some athletes take large amounts of androgen to increase muscle mass and strength. It is well-documented that testosterone, a natural circulatory androgen, administration to young, hypogonadal men and older men with lower testosterone levels stimulates muscle protein synthesis. The physiological effects of androgen administration to muscle are obvious. However, the underlying molecular mechanisms are still unknown. The myogenic regulatory factors (MRFs), including MyoD, Myf-5, myogenin, and MRF4, form a family of muscle-specific transcription factors that control skeletal muscle lineage specification and terminal differentiation. It has been reported recently that Wnt3a functions via β-catenin to induce the expression of Mox1, Pax3 and Gli2, which then activate the expression of MRFs. We hypothesized that androgen receptor (AR) may work somewhere along this pathway to regulate MRF expression and get involved in the myogenic commitment. In order to enhance the effects of AR on myogenic commitment, AR was over-expressed in cells by adenovirus and retrovirus mediated over-expression systems. C3H 10T1/2 cells was infected with adenovirus and retrovirus carrying AR cDNA, so that AR was over-expressed either transiently or permanently in cells respectively. 5-azacytidine treated C3H 10T1/2 cells have been proven to be able to switch to myoblast. Therefore, we treated the S-AR 10T1/2 cells with 5-azacytidine in the presence and absence of androgen to dissect the AR-mediated myogenesis pathway. Interestingly, 5-azacytidine treated S-AR 10T1/2 cells were able to commit to myogenic lineage in the absence of androgen with higher percentage as compared to that in the presence of androgen. In addition, flutamide, one type of anti-androgen, was unable to block the myogenic commitment in S-AR 10T1/2 cells. Hence, we suggest that AR may function in the cytoplasm to enhance myogenic commitment through either, Wnt3a-β-catenin mediated pathway or signaling pathways, or both of them.
Contents
1. Abstract in Chinese ……………………………….....I
2. Abstract in English ………………………………….II
3. List of Figures …………………………………....VIII
4. Abbreviation……………….………………………..IX
5. Introduction
5.1. Androgen and androgen receptor……………………………….1
5.2. Myogenesis and myogenic regulatory factors…………………3
5.3. Mechanisms acting upstream to MRFs………………………..5
5.4. The specific aims of this study…………………………………7
6. Materials and Methods
6.1. Cultured cells
6.1.1. Cell lines…………………………………………………9
6.1.2. Cell cultures………………………………………………9
6.2. Bacteria
6.2.1 E. coli strains…………………………………………….10
6.2.2 Propagation………………………………………………11
6.2.3 Preservation………………………………………………11
6.3. Basic cloning techniques
6.3.1 Preparation of E. coli competent cells……………………11
6.3.2 Transformation……………….…………………………...12
6.3.3 Mini-preparation …………………………………………13
6.3.4 Screening…………………………………………………14
6.3.5 Restriction Enzymes…….……………………………..…14
6.3.6 Gel extraction…………………………………………….15
6.3.7 Dephosphorylation by CIP……………………………….16
6.3.8 Ligation…………………………………………………..17
6.4. Construction of pGEMT-m4.0MyoD…………………………17
6.5. Constructions of reporter vectors driven by MyoD promoter
6.5.1 pGL3-basic-m4.0MyoD………………………………….19
6.5.2 pGL3-basic-h0.76m4.0MyoD……………………………19
6.5.3 pGL3-basic-h6.5MyoD…………………………………..20
6.6. Modification of insert and vector DNA ends
6.6.1 Klenow…………………………………………………...20
6.6.2 RT-PCR…………………………………………………..20
6.7. Generation of adenovirus
6.7.1 Adenovirus expression vectors…………………………..23
6.7.2 Generation of viral particles……………………………..23
6.7.3 Adenovirus infection…………………………………….23
6.8. Generation of retrovirus
6.8.1 Retrovirus expression vectors…………………………...24
6.8.2 Generation of retrovirus carrying hAR………………….24
6.8.3 Retrovirus infection……………………………………...25
6.9. Transfection assay……….……………………………………25
6.10. Western blots
6.10.1 Lysis of mammalian cells ………………………………26
6.10.2 Transfer of protein from SDS-Polyacrylamide gels to PVDF……………..…………………………………….27
6.10.3 Blocking binding sites and binding of 1st antibody and 2nd antibody………………………………………………….28
6.10.4 Stripping…………….…………………………………..28
7. Results ……………………………………………………………30
8. Discussion………………………………………………………..40
9. References…………………………………………………….....46
10. Figure legends…...…………………………………49
11. Figures………………...………………………………………..57
Appendix I………………….………………………………………..82
Appendix II…………….………………………………85
9. References
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