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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林甫容(Fu-Jung Lin) | |
| dc.contributor.author | Jia-Rong Yang | en |
| dc.contributor.author | 楊嘉容 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:14:01Z | - |
| dc.date.copyright | 2022-09-26 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-23 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84513 | - |
| dc.description.abstract | 轉錄因子 FOXM1 屬於 Forkhead box proteins 之一，已知能調控細胞生長、增生及分化，並高度表達於腫瘤細胞；此外，FOXM1 在哺乳動物的發育過程也扮演了重要角色。本實驗室在初步的研究結果中發現，FOXM1 高度表現於小鼠睪丸組織內的生殖細胞，且小鼠的生殖細胞特異性剔除 Foxm1 基因，便會導致其不孕，顯示 FOXM1 可能也在小鼠的精子生成過程扮演重要的角色。因此本篇研究接續探討 FOXM1 在精子生成過程中所參與的角色以及其對於減數分裂的調節機制。 我們利用重組酶 vasa-Cre 建立條件式剔除生殖細胞之 Foxm1 基因的小鼠模型，在染色體擴散分析的實驗結果中發現，Foxm1 缺失會導致生殖細胞內的性染色體聯會異常 (asynapsis)，且 DNA 延遲修復、精子生成的進程停滯於減數分裂前期 (Meiosis I - Prophase)。而以 Microarray 技術及 RT-qPCR 分析小鼠睪丸組織的基因表現，我們篩選出與精子生成相關的基因群，並透過啟動子序列分析其中的 FOXM1 的核酸結合位，找到了 FOXM1 的潛在調節標的 bromodomain testis-specific protein (Brdt)。接著我們利用基因轉殖技術將標的基因 Brdt 之啟動子序列嵌入螢光報導基因的載體，並透過報導基因的活性分析 (Reporter assay) 結果發現，當 Brdt 啟動子上的 FOXM1 核酸結合序列突變，Brdt 啟動子的轉錄活性便會顯著地下降；此外，以 FOXM1 siRNA 引發細胞內 FOXM1 基因沉默之後，不僅會造成細胞內 BRDT mRNA 的表現量減少，也同樣導致報導基因的轉錄活性下降，顯示 FOXM1 可以調節 BRDT 的轉錄活性。後續我們利用染色質免疫共沉澱技術 (Chromatin immunoprecipitation assay; ChIP) 結合 FOXM1 siRNA 及 FOXM1 的抑制劑 Siomycin A，在人類睪丸癌細胞株 NT2/D1 證明了 FOXM1 可以直接地結合至 Brdt 的啟動子區域，藉此調節 Brdt 的表現。 綜合上述研究結果，我們推論小鼠的 Foxm1 缺失對於生殖細胞在減數分裂過程所產生的不良影響，可能是由於其下游調節標的 Brdt 表現異常所導致的。這項發現或許能為不孕症的診斷或是男性避孕的新藥研發提供一個新穎的方向。 | zh_TW |
| dc.description.abstract | Transcription factor FOXM1 plays important roles in mammalian development. It involves in cell growth, proliferation and differentiation. In addition, it is highly expressed in a variety of human tumor cells. In our previous study, we found that FOXM1 is highly expressed in germ cells of mouse testis, and conditional knockout of Foxm1 in germ cells leads to the absence of sperm and results in infertility. It reveals that FOXM1 may play an important role in mouse spermatogenesis. In this study, we further investigate the molecular functions of FOXM1 during spermatogenesis in mice. We showed that Foxm1 deficiency in germ cells led to abnormal chromosome synapsis, and delayed the DNA repair process. Using gene expression profiles and FOXM1 binding motif analysis, we identified few FOXM1-regulated gene targets, including bromodomain testis-specific protein (brdt). In the reporter assay experiments, deletion or mutation of FOXM1 binding sites on the Brdt promoter significantly reduced its transcriptional activity. Moreover, we further demonstrated that FOXM1 directly bound to Brdt promoter using chromatin immunoprecipitation (ChIP) assay in human testicular embryonal carcinoma cell line NT2/D1. Together, our findings suggest that Brdt may be one of the downstream targets of FOXM1, and that the spermatogenesis defects observed in Foxm1-CKO mice might be due to the reduced expression of Brdt. Our study might provide new insights into male infertility and could lead to novel approaches to make contraception. | en |
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| dc.description.tableofcontents | 總目錄 論文口試委員審定書 I 致謝 II 中文摘要 IV ABSTRACT VI 總目錄 VII 圖目錄 XII 表目錄 XIII 第一章 緒論 1 第一節 前言 1 第二節 文獻回顧 2 一、 男性不孕症 (Male infertility) 2 二、 減數分裂 (Meiosis) 2 三、 精子生成 (Spermatogenesis) 3 四、 減數分裂前期 (Meiosis I-Prophase) 5 五、 轉錄因子 FOXM1 (Forkhead box protein M1) 7 （一） Forkhead box protein family 7 （二） FOXM1 在生物體內所扮演的角色與重要性 7 （三） FOXM1 對於基因表現的調節機制 8 （四） FOXM1 與有絲分裂和減數分裂的交互關係 8 第三節 研究動機 10 第二章 材料與方法 11 第一節 動物實驗 11 一、 建立條件式基因剔除小鼠 11 二、 基因型鑑定 11 （一） DNA 檢體採集及前處理 11 （二） 基因型鑑定 12 三、 動物犧牲及樣品保存 13 四、 基因表現量鑑定 (mRNA Expression Analysis by RT-qPCR) 13 （一） RNA 萃取與純化 (Extraction of Total RNA) 13 （二） DNA 去除 (DNase treatment) 14 （三） 將 Total RNA 反轉錄為 cDNA (Reverse Transcription) 14 （四） 即時定量聚合酶連鎖反應 (Quantitative real-time PCR) 15 五、 蛋白表現量分析 16 （一） 樣品製備 16 （二） 西方墨點法 16 1. 蛋白質電泳分離 16 2. 蛋白質轉印 16 3. Blocking 17 4. 免疫呈色 17 六、 染色體擴散 (Chromosome Spresd) 18 第二節 細胞實驗 19 一、 細胞培養 19 （一） 細胞培養基製備 19 （二） 細胞繼代 19 二、 建構載體 (Construction of plasmids) 20 （一） 轉型作用 (Transformation) 20 （二） 菌株培養 20 （三） 純化質體 (plasmid DNA extraction) 20 （四） 質體鑑定 (Restriction enzyme digestion) 20 （五） 重組質體的建構 22 1. 查找 cDNA 序列、基因啟動子序列 22 2. 查找 mRNA 序列 22 3. 轉錄因子結合位點分析 22 4. 野生型小鼠睪丸組織之基因體純化 (Genomic DNA Purification) 22 5. 擴增標的基因的啟動子序列 (PCR) 23 6. PCR 產物萃取 (Gel extraction) 24 7. TA Cloning 24 8. 建構重組質體 27 （六） 突變型重組質體的建立 30 1. Site Directed Mutagenesis 30 2. Truncation Mutagenesis 32 三、 螢光報導基因分析技術 (Reporter gene assay technology) 34 （一） 瞬時轉染 (Transient transfection) 34 （二） 報導基因活性檢測 (Dual Luciferase assay) 34 1. 樣品前準備 34 2. 報導基因活性檢測 34 3. 相對活性校正 35 四、 RNA interference (RNAi) 35 五、 染色質免疫沉澱分析 (Chromatin immunoprecipitation assay) 36 （一） 染色質固定 (Formaldehyde cross-link) 36 （二） 染色質片段化 (Chromatin shearing) 36 1. 樣品前處理 36 2. 超聲波震盪 36 3. 鑑定超音波處理的產物片段大小 37 （三） 免疫沉澱 (Chromatin immunoprecipitation) 37 （四） Real-time qPCR 定量分析 38 1. ChIP-qPCR 38 2. Fold Enrichment Method 38 第三章 結果與討論 39 第一節 以動物模式探討 FOXM1 轉錄因子對精子生成之重要性 39 一、 條件式剔除小鼠生殖細胞之 Foxm1 基因對其睪丸組織的表徵影響 39 二、 條件式剔除小鼠生殖細胞之 Foxm1 基因對睪丸組織的 mRNA／蛋白表現量影響 40 三、 條件式剔除小鼠生殖細胞之 Foxm1 基因對減數分裂進程之影響 41 （一） Foxm1-CKO meiocytes 之體染色體破碎且性染色體聯會異常 41 （二） Foxm1-CKO meiocytes 之體染色體 DNA 修復異常 44 （三） Foxm1-CKO 小鼠之精子生成作用停滯於 Leptotene stage 45 第二節 以細胞模式探討 FOXM1 對減數分裂的調節機制 47 一、 轉錄因子 FOXM1 可調節與減數分裂相關的功能性基因之轉錄作用 47 （一） 轉錄因子 FOXM1 之潛在調節標的基因分析 47 （二） 轉錄因子 FOXM1 之潛在調節標的基因篩選 49 （三） 轉錄因子 FOXM1 與預測標的基因啟動子之結合位點分析 49 二、 條件式剔除小鼠生殖細胞之 Foxm1 基因可誘導 Brdt 及 Setx 表現下降 53 三、 轉錄因子 FOXM1 與 Brdt 及 Setx 啟動子序列之交互關係 54 （一） 透過基因工程將標的基因的啟動子序列嵌入帶有螢光報導基因的質體 54 （二） 細胞內過量表現 FOXM1 對潛在標的基因之啟動子的活性影響 56 （三） 標的基因啟動子之 FOXM1 結合位點突變／截斷對基因轉錄活性之影響 58 四、 以人類細胞探討 FOXM1 轉錄因子對 Brdt 之調節機制 61 （一） HEK293T 細胞轉染 FOXM1 siRNA 後不同時間點的 mRNA 表現量 61 （二） HEK293T 細胞轉染 FOXM1 siRNA 對 BRDT promoter 的活性影響 63 （三） NT2/D1 細胞轉染 FOXM1 siRNA 後不同時間點的 mRNA 表現量 64 （四） NT2/D1細胞轉染 FOXM1 siRNA 對 Brdt promoter 的活性影響 66 五、 以染色質免疫沉澱 (ChIP) 證實 FOXM1 直接地結合至 BRDT promoter 68 （一） NT2/D1 細胞處理 FOXM1 抑制劑—Siomycin A之 ChIP 實驗結果 68 （二） NT2/D1 細胞處理 FOXM1 siRNA 之 ChIP 實驗結果 71 第三節 討論 73 一、 特異性剔除生殖細胞之 Foxm1 對小鼠睪丸組織 FOXM1 蛋白表現的影響 73 二、 Foxm1 基因剔除導致生殖細胞內的 X-Y Pairing 失常並阻斷精子生成 73 三、 Foxm1 基因缺陷使減數分裂的過程滯留於 Leptotene stage 的原因 74 四、 FOXM1 轉錄因子在哺乳動物生殖細胞內調節減數分裂的機制 77 （一） Brdt 與 FOXM1 的關聯性 78 （二） Setx 與 FOXM1 的關聯性 78 （三） FOXM1 與 Brdt promoter 及 Setx promoter 的交互作用 79 1. FOXM1 可以增強 Brdt / Setx promoter 對報導基因的轉錄活性 79 2. Brdt 和 Setx 啟動子上的 FOXM1 結合位點是活化轉錄作用的關鍵 80 3. Setx-promoter 的活性可能不是專一性地受到 FOXM1 所調控 80 （四） FOXM1 直接地調節 Brdt 基因的轉錄效率 81 1. Brdt 在轉錄起始點上游 -1036 / -927 的區域帶有 FKH motif 81 2. FOXM1-deletion 對減數分裂造成的不良影響可能與 Brdt 表現異常有關 81 （五） FOXM1 與減數分裂相關的其他潛在調節標的 82 第四章 結論 84 第五章 研究展望 85 第六章 附錄 86 第一節 基因轉殖質體序列 86 一、 pGL3-Basic-Brdt 1.5k promoter (6362 bp) (-,+) 86 二、 pGL3-Basic-Brdt 916 bp promoter (5778 bp) (+,+) 88 三、 pGL3-Basic-Brdt 465 bp promoter (5327 bp) (-,+) 89 四、 pGL3-Basic-mSetx 2.8k promoter (7736 bp) (-,+) 91 五、 pGL3-Basic-mSetx 2141 bp promoter (7003 bp) (-,+) 93 六、 pGL3-Basic-mSetx 1k promoter (5862 bp) 95 第二節 精子生成異常與不孕症之遺傳研究報告58 98 第七章 參考文獻 102 | |
| dc.language.iso | zh-TW | |
| dc.title | 利用轉錄因子 FOXM1 基因剔除小鼠探討其在精子生成之角色 | zh_TW |
| dc.title | Characterization of the Roles of FOXM1 During Spermatogenesis in Mice | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡沛學(Pei-Shiue Tsai),王翊青(I-Ching Wang),林盈宏(Ying-Hung Lin) | |
| dc.subject.keyword | 男性不孕,精子生成,減數分裂,轉錄因子, | zh_TW |
| dc.subject.keyword | Male infertility,Spermatogenesis,Meiosis,Transcription factor, | en |
| dc.relation.page | 107 | |
| dc.identifier.doi | 10.6342/NTU202203727 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-09-24 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2024-08-30 | - |
| 顯示於系所單位： | 生化科技學系 | |
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