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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊雅倩(Ya-Chien Yang) | |
dc.contributor.author | I-Chien Lin | en |
dc.contributor.author | 林宜芊 | zh_TW |
dc.date.accessioned | 2021-06-16T09:15:51Z | - |
dc.date.available | 2025-08-14 | |
dc.date.copyright | 2020-08-27 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59084 | - |
dc.description.abstract | 大腸直腸癌在世界癌症死亡率排名位於前三名。本實驗室先前研究提出位於人類第三號染色體3p26.3 之Contactin 4 (CNTN4) 於大腸直腸癌可能扮演抑癌基因的角色。於HCT116細胞株表現CNTN4可抑制細胞增生、固著及非固著依賴性胞落形成的能力;在活體動物實驗中,則顯示CNTN4可抑制HCT116細胞株於裸鼠皮下腫瘤的生成及血管新生;針對抑癌機轉初步推測CNTN4可與Receptor-type protein tyrosine phosphatase gamma (PTPRG)交互作用,進而抑制ERK1/2及其下游CREB之磷酸化,並使PGC-1α表現下降,影響粒線體生合成及降低粒線體DNA拷貝數。本論文為進一步了解CNTN4與PTPRG之相互作用,首先發現表現CNTN4可增加PTPRG蛋白表現。為確認以HCT116/CNTN4穩定表現細胞株所觀察到的抑癌機制,本論文藉由慢病毒轉導CNTN4質體於其他大腸直腸癌細胞株,並建立HCT15/CNTN4及COLO205/CNTN4穩定表現細胞株。藉由分離細胞膜蛋白及細胞質蛋白,發現過表現CNTN4可表現至細胞膜,然而,大部分為CNTN4-V5蛋白表現於細胞質。於HT29、SW620、HCT15及COLO205大腸直腸癌細胞表現CNTN4後皆可抑制細胞增生。於HCT15細胞表現CNTN4可降低CREB及其上游蛋白ERK1/2之磷酸化,下游路徑之PGC-1α表現量亦隨之下降,亦可抑制粒線體DNA拷貝數及粒線體質量;然而,於COLO205細胞表現CNTN4僅可降低CREB之磷酸化和PGC-1α表現量,以及抑制粒線體質量,因於此細胞之上游調控路徑須進一步研究。於臨床檢體檢測CNTN4表現與粒線體DNA含量,發現於大腸直腸癌腫瘤部分CNTN4表現量與粒線體DNA表現量呈現負相關趨勢。因先前利用人類磷酸化蛋白激酶晶片發現HCT116 細胞表現CNTN4後可降低c-Jun磷酸化,目前已知c-Jun受ERK1/2、JNK及p38等MAPK家族蛋白調控,故於HCT116/CNTN4細胞確認c-Jun及其上游分子ERK1/2磷酸化程度下降。已知c-Jun參與細胞凋亡及細胞增生的調控路徑,故進一步確認HCT116/CNTN4細胞的Bcl-2蛋白表現下降,並增加cleaved caspase 3生成,而於HCT116/CNTN4細胞之自發性細胞凋亡增加;當利用H2O2處理HCT116和HCT15細胞,表現CNTN4可促進細胞凋亡,據此推測HCT116細胞表現CNTN4可減少ERK1/2及c-Jun磷酸化,進而抑制Bcl-2表現和增加活化態caspase 3,因而可增加細胞凋亡比例。綜合上述,CNTN4會與PTPRG交互作用影響下游pERK/pCREB/PGC-1α路徑,進而改變粒線體生合成;同時調控pERK/p-c-Jun/Bcl-2路徑,促進細胞凋亡。 | zh_TW |
dc.description.abstract | Colorectal cancer (CRC) is the third leading cause of cancer death in the world. Our previous study has identified that Contactin 4 (CNTN4) located at human chromosome 3p26.3 plays an important role as a tumor suppressor gene in CRC. Ectopic expression of CNTN4 in HCT116 cells could inhibit cell proliferation, anchorage-dependent and anchorage-independent colony formation in vitro. Furthermore, CNTN4 expression could inhibit the subcutaneous xenograft tumor formation and angiogenesis in nude mice. The mechanism study of CNTN4 indicated that CNTN4 could interact with protein tyrosine phosphatase γ (PTPRG), and then inhibit the phosphorylation of ERK1/2 and its downstream protein, including phospho-CREB and PGC-1α, resulting in the modulation of energy metabolism. CNTN4 would not only promote mitochondrial fission, but also decrease mitochondrial DNA copy number and mitochondrial mass via interaction between PGC-1α and TFAM. In the study, to establish more CRC cell models, lentiviral transduction was conducted to transiently express CNTN4 in HCT15 and SW620 cells. At the same time, I established HCT15/CNTN4, COLO205/CNTN4 and SW620/CNTN4 single stable transfectants. I first showed that overexpressed CNTN4 could present on the membrane, but still mainly in the cytosol. Next, I verified that ectopic expression of CNTN4 in HCT116, HCT15 and COLO205 cells could increase expression of PTPRG protein. CNTN4 expression inhibited cell proliferation in all of transient CNTN4 expression cell lines and single stable clones. Meanwhile, CNTN4 expression decreased mitochondrial DNA copy number and mitochondrial mass via pERK/pCREB/PGC-1α pathway in HCT15 cells, which is consistent with the results observed in HCT116/CNTN4 stable clones. However, CNTN4 expression in COLO205 cells could only downregulate pCREB, PGC-1α, and mitochondrial mass. Therefore, the other molecules involved in the mechanism should be further addressed. In the 50 paired CRC tissues, there was a negative trend between relative CNTN4 expression and mitochondrial DNA copy number. On the other hand, CNTN4 expression in HCT116 cells could reduce c-Jun phosphorylation compared with HCT116/Mock by using Human Phospho-Kinase array. We further confirmed the downregulation of pERK1/2 and phospho-c-Jun in HCT116/CNTN4 transfectants. It is well known that c-Jun can regulate cell proliferation and apoptosis. Therefore, we determined the expression of Bcl-2 and cleaved caspase 3. Ectopic expression of CNTN4 in HCT116 cells indeed downregulated Bcl-2 and increased active caspase 3. As expected, CNTN4 expression could increase spontaneous apoptosis in HCT116 cells and H2O2-induced apoptosis in HCT116 and HCT15 cells, suggesting that CNTN4 might promote apoptosis through pERK/p-c-Jun/Bcl-2 pathway. In conclusion, CNTN4 expression could interact with PTPRG to mediate mitochondrial biogenesis through pERK/pCREB/ PGC-1α pathway and promote apoptosis through pERK/p-c-Jun/Bcl-2 pathway.
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dc.description.provenance | Made available in DSpace on 2021-06-16T09:15:51Z (GMT). No. of bitstreams: 1 U0001-1408202016342200.pdf: 5671993 bytes, checksum: a100ebaf93673c41ffe56344601ab3ce (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 誌謝 i 摘要 ii 英文摘要 iv 縮寫對照表 vi 圖目錄 xiv 表目錄 xv 一、研究背景 1 1. 大腸直腸癌 1 1.1 簡介 1 1.2腫瘤生成機制 2 1.3 大腸直腸癌分子特徵與標靶藥物的綜合分類系統 3 1.4 癌症分期 5 2. Contactin 4 (CNTN4) 9 2.1 Contactin家族與CNTN4簡介 9 2.2 CNTN4相關研究 10 2.3 CNTN4與癌症相關研究 11 3. Protein Tyrosine Phosphatase Receptor gamma (PTPRG) 13 3.1 PTPRG簡介 13 3.2 PTPRG與癌症相關研究 13 3.3 RPTP與Contactin相關研究 14 4. 細胞凋亡 15 4.1 細胞凋亡起始 15 4.2細胞凋亡過程 15 4.3抑制細胞凋亡之分子 15 5. 粒線體動態平衡 16 5.1 粒線體融合 16 5.2 粒線體分裂 17 5.3 粒線體生合成 18 6. 實驗室先前CNTN4相關研究 19 6.1 第三號染色體失異合性檢測 19 6.2 CNTN4抑癌功能之研究 19 6.3 CNTN4抑制大腸直腸癌細胞表現促血管新生因子 urokinase-type plasminogen activator (uPA) 之研究 19 6.4 溶解型CNTN4抑癌功能之研究 20 6.5 CNTN4抑制大腸直腸癌細胞粒線體能量代謝之研究及其機制 20 二、研究目標 21 三、材料與方法 22 1. 細胞培養 22 1.1 細胞株 22 1.2 細胞培養 22 2. 利用siRNA抑制CNTN4表現 23 2.1試劑清單 23 2.2 siPTPRG 24 3. 慢病毒轉導lentivirus transduction 24 3.1 暫時性表現細胞株 24 3.2 穩定表現細胞株篩選 25 4. DNA 萃取 25 5. RNA 萃取 26 6. 反轉錄合成互補 DNA 26 7. 即時定量聚合酶連鎖反應 27 8. 蛋白質的抽取及定量 27 9.細胞膜蛋白萃取 28 10. 西方墨點法 28 10.1 蛋白質檢測 28 10.2 抗體清單 29 11. 細胞增生分析 30 11.1 黏附型細胞 30 11.1 懸浮型細胞 31 12. 細胞凋亡分析 31 13. 粒線體形態與總量分析 32 14. 公開資料庫之數據分析 32 15. 統計 32 四、研究結果 33 1. 於HCT15、COLO205和SW620培養CNTN4穩定表現細胞株 33 2. 探討HCT116/CNTN4穩定表現細胞株細胞膜蛋白及細胞質蛋白之CNTN4表現量 33 3. 探討大腸直腸癌細胞表現CNTN4對於PTPRG 表現之調控 33 4. 利用慢病毒轉導使大腸直腸癌細胞株HT29和SW620暫時性表現CNTN4及觀察其調控機制 34 5. 利用慢病毒轉導使大腸直腸癌細胞株HCT15、HT29和SW620暫時性表現CNTN4對於細胞增生之影響 35 6. 於HCT15/CNTN4、COLO205/CNTN4穩定表現細胞株觀察CNTN4對細胞增生之影響 35 7. 利用HCT15/CNTN4和COLO205/CNTN4穩定表現細胞株觀察其抑癌機制 36 8. 利用粒線體螢光染色探討CNTN4表現造成HCT15、COLO205和SW620細胞粒線體基因之改變 36 9. 利用即時定量聚合酶連鎖反應探討CNTN4表現造成HCT15、COLO205和SW620細胞粒線體基因變化 37 10. 利用即時定量聚合酶連鎖反應探討粒線體基因於大腸直腸癌病患腫瘤之表現及其與CNTN4表現量之相關性 38 11. 探討於HCT116表現CNTN4對於磷酸化蛋白激酶及其上游路徑之調控 39 12. 探討於HCT116細胞表現CNTN4對c-Jun調控Bcl-2及其下游caspase 3和PARP-1變化 39 13. 探討於HCT116及HCT15細胞表現CNTN4對細胞凋亡的影響 40 14. 探討於HCT116、HCT15及COLO205細胞表現CNTN4後所調控之抑癌機轉 41 五、討論 42 圖 48 表 69 參考文獻 77 附錄 84 | |
dc.language.iso | zh-TW | |
dc.title | 探討 Contactin 4 於大腸直腸癌之抑癌分子機轉 | zh_TW |
dc.title | Study of contactin 4-mediated molecular mechanism in colorectal cancer | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇剛毅(Kang-Yi Su),郭靜穎(Ching-Ying Kuo),饒梓明(Tzu-Ming Jao) | |
dc.subject.keyword | 大腸直腸癌,Contactin 4,PTPRG,CREB磷酸化,c-Jun磷酸化,粒線體生合成,細胞凋亡, | zh_TW |
dc.subject.keyword | Colorectal cancer,Contactin 4 (CNTN4),Protein tyrosine phosphatase γ (PTPRG),CREB phosphorylation,c-Jun phosphorylation,apoptosis,mitochondrial biogenesis, | en |
dc.relation.page | 85 | |
dc.identifier.doi | 10.6342/NTU202003449 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-15 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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