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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡孟勳 | zh_TW |
| dc.contributor.advisor | Mong-Hsun Tsai | en |
| dc.contributor.author | 徐資恆 | zh_TW |
| dc.contributor.author | Tzu-Heng Hsu | en |
| dc.date.accessioned | 2025-08-18T01:03:48Z | - |
| dc.date.available | 2025-08-18 | - |
| dc.date.copyright | 2025-08-15 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-06 | - |
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Journal of pharmacological and toxicological methods, 94, 26-33. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/98608 | - |
| dc.description.abstract | 肺癌是一種最常見且致死率極高的癌症，在全球被視為最具挑戰性的公共衛生問題。儘管已有新的治療方法被開發出來，但腫瘤的多樣性和對治療的抗性等問題仍有待克服。因此，更深入地了解肺癌細胞中的分子調控機制，將有助於我們更全面地認識疾病的運作及進展，從而為未來的研究提供重要的見解。我們先前的研究顯示，Semaphorin 6A（SEMA6A）在肺癌組織及細胞株中的表現量顯著降低，而過表達 SEMA6A full-length（SEMA6A-FL）可顯著誘導肺癌細胞凋亡。當SEMA6A-FL失去SEMA域時，其胞內區域會與FADD（Fas-associated death domain）結合，進而透過裂解Caspase-8誘導細胞凋亡。然而，即使SEMA6A-FL在肺癌細胞中表現後可觀察到約10%的凋亡比例，其胞內區域卻並未透過FADD和Caspase-8誘導此過程。此外，先前研究發現SEMA6A-FL可誘發內質網壓力，因此我們推測SEMA6A-FL是透過內源性凋亡途徑引發細胞死亡。早期研究也顯示，SEMA6A-FL可在細胞中以單體和二聚體的形式存在，並且SEMA6A-FL透過其SEMA域中第415位胺基酸形成二聚體，進而與Plexin-A2相互作用。這些結果表明，SEMA6A-FL的單體和二聚體可能具有不同的生物學功能。因此，我們假設SEMA6A-FL的結構，尤其是其形成二聚體的能力，可能會顯著影響其誘導內源性細胞凋亡的機制。在本研究中，我們確認了兩個特定的突變——M415C（促進二聚化）和I322E（減少二聚化）——可以有效改變SEMA6A-FL的二聚化能力。為了探討這些結構對細胞功能的影響，我們進行了多項實驗，包括細胞生長測試、集落形成存活分析、細胞凋亡檢測、細胞週期分析以及凋亡相關蛋白的表現量測定。結果顯示，當 SEMA6A-FL的二聚化能力被減弱時，細胞生長或凋亡與對照組相比並無顯著變化。然而，當SEMA6A-FL的二聚化能力增強時，肺癌細胞的生長顯著減少，且凋亡顯著增加。此外，我們製備了更容易形成二聚體的SEMA6A-FL截短版本，並將這些版本過表現於肺癌細胞中，結果顯示細胞生長顯著減少且凋亡增加，進一步強調了 SEMA6A-FL二聚化在誘導細胞凋亡中的重要角色。另外，增加SEMA6A-FL二聚體會增強肺癌細胞內質網壓力的程度，而且，下游Caspase-9的裂解產物也跟著增加。這代表SEMA6A-FL是透過二聚體結構引起內質網壓力，接著誘發肺癌細胞的內源性凋亡訊號。 | zh_TW |
| dc.description.abstract | Lung cancer is a common and deadly disease, representing a significant challenge to public health worldwide. Despite new therapies being developed, problems like tumor diversity and resistance to treatment still need to be overcome. Therefore, to better understand the molecular regulatory mechanisms in lung cancer cells will help us learn more about how the disease works and progress, leading to important insights for future research. Our previous research demonstrated that Semaphorin 6A (SEMA6A) expression is significantly reduced in lung cancer tissues and cell lines, while overexpression of SEMA6A-FL can trigger apoptosis significantly in the lung cancer cell lines. Although the intracellular domain of SEMA6A-FL does not interact with FADD, it has been shown to induce endoplasmic reticulum (ER) stress in lung cancer cells, suggesting that SEMA6A-FL may trigger apoptosis through the intrinsic pathway. Additionally, earlier studies have found that SEMA6A-FL exists in both monomer and dimer forms in the cells. It has also been shown that SEMA6A-FL dimerizes through a specific amino acid (415th position in the SEMA domain), which allows it to interact with another protein called Plexin-A2. These findings suggest that the monomer and dimer forms of SEMA6A-FL might have different biological functions. Based on this, we hypothesize that the structure of SEMA6A-FL, especially its ability to form dimers, may affect how it triggers intrinsic apoptosis. In the study, we confirmed that two specific mutations—M415C (which promotes dimerization) and I322E (which reduces dimerization)—can effectively change how SEMA6A-FL dimerizes. To see how these structures impact cell functions, we carried out several experiments, such as testing cell growth, clonogenic survival, apoptosis detection, cell cycle analysis, and measuring proteins involved in apoptosis. The results showed that when dimerization of SEMA6A-FL was reduced, there was no significant change in cell growth or apoptosis compared to the control group. However, when dimerization of SEMA6A-FL was increased, cell growth was significantly reduced, and apoptosis was greatly increased in the lung cancer cells. We also created truncated versions of SEMA6A-FL that are more likely to dimerize, and overexpressing these versions in lung cancer cells caused a significant reduction in cell growth and an increase in apoptosis, further highlighting the role of SEMA6A-FL dimerization in inducing cell death. Moreover, an increase in SEMA6A-FL dimerization enhances the degree of ER stress, accompanied by an increase in the cleavage products of downstream Caspase-9. This suggests that SEMA6A-FL induces ER stress through its dimeric structure, thereby triggering the intrinsic apoptotic signaling pathway in lung cancer cells. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-18T01:03:48Z No. of bitstreams: 0 | en |
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| dc.description.tableofcontents | 摘要 I
Abstract III 目次 V 表次 XI 圖次 XII 第一章、 文獻回顧與研究背景 1 1.1、 肺癌的全球挑戰與臨床現狀 1 1.2、 肺癌在台灣的流行病學特徵 2 1.3、 肺癌的致病原因 3 1.4、 肺癌的分類 4 1.4.1、 肺腺癌（Adenocarcinoma） 4 1.4.2、 鱗狀細胞癌（Squamous Cell Carcinoma） 4 1.4.3、 大細胞癌（Large Cell Carcinoma） 5 1.4.4、 小細胞肺癌（SCLC） 5 1.5、 肺癌治療的挑戰 5 1.6、 目前肺癌分子機制的治療策略 6 1.7、 探索肺癌潛在的未知分子機制 6 第二章、 SEMA6A與細胞凋亡的研究背景 8 2.1、 Semaphorin蛋白質家族 8 2.2、 SEMA6A的生物學背景及其在癌症中的角色 9 2.2.1、 SEMA6A的研究背景 9 2.2.2、 SEMA6A在肺癌中扮演的角色 10 2.3、 細胞凋亡（Apoptosis）的訊號途徑 14 2.3.1、 Extrinsic Apoptosis Pathway（外源性細胞凋亡途徑） 14 2.3.2、 Intrinsic Apoptosis Pathway（內源性細胞凋亡途徑） 14 第三章、 研究動機與目的 16 3.1、 SEMA域參與SEMA6A二聚化 16 3.2、 探討SEMA6A-FL誘導的凋亡路徑 19 3.3、 研究目的 21 第四章、 材料與方法 23 4.1、 細胞培養（Cell Culture） 23 4.1.1、 製備培養基（Medium Preparation） 23 4.1.2、 細胞株（Cell Lines） 24 4.1.3、 細胞繼代（Cell Subculture） 24 4.2、 重組質體的製作方法（Cloning of Recombinant Plasmids） 25 4.2.1、 製作SEMA6A的截短片斷（Truncated SEMA6A Amplification） 25 4.2.2、 SEMA6A-FL點突變（Point Mutation of SEMA6A-FL） 26 4.2.3、 連接領導胜肽（Signal Peptide Elongation） 27 4.2.4、 雙重限制酶消化（Double Digestion Using Restriction Enzymes） 28 4.2.5、 連接反應（Ligation） 29 4.2.6、 細胞轉型（Transformation） 30 4.2.7、 質體製備（Plasmids Preparation） 30 4.2.8、 Agarose gel、LB agar和LB broth的製備 31 4.3、 慢病毒轉染系統（Lentivirus Transduction System） 32 4.3.1、 病毒製備（Viruses Production） 32 4.3.2、 細胞感染（Cell Infection） 32 4.4、 mRNA基因表達分析（mRNA Expression Analysis） 33 4.4.1、 RNA萃取（RNA Extraction） 33 4.4.2、 反轉錄聚合酶連鎖反應（Reverse Transcription PCR） 34 4.4.3、 及時定量聚合酶連鎖反應（Quantitative Real-Time PCR, qPCR） 35 4.5、 蛋白質表達分析（Protein Expression Analysis） 36 4.5.1、 蛋白質萃取（Protein Extraction） 36 4.5.2、 蛋白質定量（Protein Quantification） 36 4.5.3、 蛋白質變性（Protein Denaturation） 36 4.5.4、 SDS-PAGE（Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis） 37 4.5.5、 西方免疫點墨法（Western Immunoblotting） 38 4.5.6、 製備SDS-PAGE緩衝液（Preparation of SDS-PAGE Buffer） 38 4.6、 細胞功能分析（Cell Functional Analysis） 39 4.6.1、 細胞增殖實驗（Cell Proliferation Assay） 39 4.6.2、 克隆形成實驗（Clonogenic Assay） 39 4.6.3、 XBP1剪切實驗（XBP1 Splicing Assay） 40 4.6.4、 細胞遷移實驗（Migration Assay Using Transwell） 41 4.7、 流式細胞儀分析（Flow Cytometry Analysis） 41 4.7.1、 雙重胸腺嘧啶阻斷（Double Thymidine Blocking） 41 4.7.2、 細胞週期實驗（Cell Cycle Assay） 42 4.7.3、 細胞凋亡實驗（Apoptosis Assay） 42 4.8、 統計分析（Statistical Analysis） 43 第五章、 實驗結果 44 5.1、 成功構建SEMA6A-FL M415C及I322E點突變體 44 5.2、 SEMA6A-FL M415C和I322E分別增強與減弱蛋白質二聚化的能力 44 5.3、 SEMA6A-FL二聚體抑制肺癌細胞的生長能力 45 5.4、 SEMA6A-FL的結構改變不顯著影響細胞週期進展 45 5.5、 SEMA6A-FL透過二聚體誘導細胞凋亡 46 5.6、 成功構建二種與二聚化相關的SEMA6A截短變異體 46 5.7、 二種SEMA6A截短變異體皆具增強其二聚化能力 47 5.8、 過表達二種SEMA6A截短變異體皆顯著抑制肺癌細胞的生長活性 47 5.9、 二種SEMA6A截短變異體均不顯著影響細胞週期進展 47 5.10、 過表達二種SEMA6A截短變異體皆顯著促進細胞凋亡 48 5.11、 SEMA6A-FL結構改變不影響Caspase-8裂解活性 48 5.12、 SEMA6A-FL二聚體增加內質網壓力及Caspase-9裂解產物 49 5.13、 缺失SEMA6A 365-462胺基酸片段顯著抑制肺癌細胞的遷移能力 49 第六章、 討論 50 6.1、 SEMA6A點突變於肺癌細胞凋亡功能中的探討 50 6.1.1、 SEMA6A-FL單體與二聚體間可能存在結構動態平衡 50 6.1.2、 SEMA6A-FL是否正確定位於細胞膜尚待驗證 50 6.1.3、 SEMA6A-FL二聚體提升ER Stress並促進Caspase-9裂解 50 6.1.4、 SEMA6A-FL與6Acyto和6AΔsema誘導之凋亡路徑存在差異 51 6.1.5、 SEMA6A-FL M415C誘導凋亡之訊號傳遞方向仍待明確釐清 51 6.2、 對內源性（粒線體依賴性）凋亡路徑的討論 52 6.2.1、 需謹慎評估過表達基因所致ER Stress之實驗性假象可能性 52 6.2.2、 探討造成Mitochondrial Damage的其他原因 53 6.3、 提出蛋白質功能調控的新解釋機制 54 6.4、 SEMA6A截短體對肺癌細胞凋亡及細胞遷移的討論 55 6.4.1、 SEMA6A截短體之功能驗證顯示其促進二聚化並誘導凋亡 55 6.4.2、 SEMA6A二聚體形成可能受其他結構區域調控 55 6.4.3、 SEMA6A第365-462位胺基酸片段可能參與肺癌細胞遷移機制 56 第七章、 未來研究方向 57 7.1、 探討SEMA6A-FL誘導內源性凋亡過程中Cytochrome c的釋放 57 7.2、 探討其他造成Mitochondrial Damage的原因 57 7.3、 製備高純度SEMA6A-FL單體與二聚體 58 7.4、 利用純化SEMA6A-FL單體與二聚體比較其細胞功能 58 第八章、 結論 59 實驗結果圖 60 引子序列表 81 參考資料 83 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | Semaphorin 6A膜蛋白 | zh_TW |
| dc.subject | 肺癌細胞 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 蛋白質單體 | zh_TW |
| dc.subject | 蛋白質二聚體 | zh_TW |
| dc.subject | protein dimer | en |
| dc.subject | protein monomer | en |
| dc.subject | apoptosis | en |
| dc.subject | Semaphorin 6A | en |
| dc.subject | Lung cancer cells | en |
| dc.title | 探討Semaphorin 6A二聚化在誘導肺癌細胞內源性凋亡中的功能 | zh_TW |
| dc.title | To Study the Functions of Semaphorin 6A Dimerization in Inducing Intrinsic Apoptosis in Lung Cancer Cells | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 賴亮全;陳立涵 | zh_TW |
| dc.contributor.oralexamcommittee | Liang-Chuan Lai;Li-Han Chen | en |
| dc.subject.keyword | 肺癌細胞,Semaphorin 6A膜蛋白,蛋白質二聚體,蛋白質單體,細胞凋亡, | zh_TW |
| dc.subject.keyword | Lung cancer cells,Semaphorin 6A,protein dimer,protein monomer,apoptosis, | en |
| dc.relation.page | 93 | - |
| dc.identifier.doi | 10.6342/NTU202503246 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-08-09 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 生物科技研究所 | - |
| dc.date.embargo-lift | 2025-08-18 | - |
| 顯示於系所單位： | 生物科技研究所 | |
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