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研究生:孫亞筠
研究生(外文):SUN, YA-YUN
論文名稱:探討CXCR4調控肝細胞癌幹細胞的自我新生、增生、侵襲能力及形成血管生成擬態所扮演之角色
論文名稱(外文):To Explore the Role of CXCR4 in Regulating Self-renewal, Proliferation, Invasion and Vasculogenic Mimicry From Hepatocellular Carcinoma Cancer Stem Cell
指導教授:陳至理
指導教授(外文):CHEN,CHIH-LI
口試委員:吳肇卿王啟仲
口試日期:2014-01-23
學位類別:碩士
校院名稱:輔仁大學
系所名稱:基礎醫學研究所碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:69
中文關鍵詞:肝細胞癌癌幹細胞趨化因子接受器
外文關鍵詞:HCCCancer stem cellCXCR4
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肝細胞癌具有高度血管新生的現象,而腫瘤內血管新生的機制尚需釐清。近年來許多研究指出除了血管增生外,癌幹細胞可轉型成內皮細胞或類血管平滑肌細胞而形成血管生成擬態(vasculogenic mimicry,簡稱VM)並達到腫瘤血管形成的效果。而腫瘤血管生成擬態被認為是惡性腫瘤細胞新生成且具有功能性的血管構造,用來提供腫瘤快速的生長。已知CXCR4及其配位子CXCL12在血管增生的過程中扮演重要角色,然而CXCR4在調節癌幹細胞形成血管生成擬態中所扮演的角色尚不清楚,因此本論文探討CXCR4調控肝細胞癌幹細胞形成血管生成擬態所扮演之角色。本論文選擇具有高度癌幹細胞特性的肝細胞癌細胞(Mahlavu細胞),我們利用慢病毒(lentivirus)的感染技術將CXCR4 shRNA送入Mahlavu細胞中抑制CXCR4的表現,反之,利用脂小體(liposome)的轉染技術將pCAG-CXCR4-lacZ質體送入Mahlavu細胞中使CXCR4過度表現。接著分別測試CXCR4的改變對細胞產生的影響。我們發現抑制CXCR4的表現後,Mahlavu細胞自我新生的能力受到抑制,而細胞的增生以及侵襲轉移的能力也同樣受到抑制,結果顯示改變CXCR4會造成Mahlavu在腫瘤幹細胞特性上的改變。反之,過度表現CXCR4則會促進Mahlavu細胞自我新生能力和侵襲轉移能力的增加。另外,經由皮下注射經處理的Mahlavu細胞至NOD-SCID小鼠,實驗結果指出抑制CXCR4會抑制Mahlavu細胞在NOD-SCID小鼠上的腫瘤生長能力。而in vitro的血管生成實驗亦發現抑制CXCR4會抑制VM的形成,同時也發現抑制CXCR4後也減少了VM形成之相關基因的表現,例如VE-cadherin、EPHA2等。因此,CXCR4能調控Mahlavu細胞的癌幹細胞特性,如自我新生、增生、侵襲轉移和腫瘤形成,並能透過VE-cadherin、EPHA2的路徑參與血管生成擬態的過程。
Hepatocellular carcinoma (HCC) is highly vascularized. The exact mechanism underlying vascularization still needs to be unraveled. In recent years, many studies have indicated that in addition to angiogenesis, cancer stem cells (CSCs) are capable of forming functional blood vessels de novo by transdifferentiating into endothelial progenitor cells, endothelial cells, or vascular smooth muscle-like cells (called vasculogenic mimicry). Tumor cell vasculogenic mimicry (VM) describes the functional plasticity of aggressive cancer cells forming de novo vascular networks, thereby providing a perfusion pathway for rapidly growing tumors. CXCR4 and its ligand CXCL12 play an important role in the angiogenesis, however the role of CXCR4 in regulating the vasculogenic mimicry(VM) from hepatocellular carcinoma cancer stem cell is unclear. The aim of this study is to explore the role of CXCR4 in regulating the VM from hepatocellular carcinoma cancer stem cell. In this study the Mahlavu cell, a HCC cell line, was chosen to perform the experiments because this cell line has high characteristics of cancer stem cell. We knockdowned the expression of CXCR4 using shRNA delivered by lentivirus infection in Mahlavu cells. The plasmid, pCAG-CXCR4-lacZ, was transfected into Mahlavu cell to constitutively expressing CXCR4.We demonstrated that a knockdown of CXCR4 results in significantly reduced self-renewal, proliferation, invasiveness and tumorigenesis of Mahlavu cell. Conversely, overexpression of CXCR4 remarkably enhanced self-renewal and invasion. Using in vitro tube formation assay, we showed that knockdown of CXCR4 reduced tube formation of Mahlavu cell. Furthermore, knockdown of CXCR4 also reduced the expression of VM-associated markers. Therefore, CXCR4 can regulate the characteristics of cancer stem cells in Mahlavu, such as self-renewal, proliferation, invasion and tumorigenesis. More importantly, CXCR4 may participate in VM formation through regulating the expression of VE-cadherin and EPHA2.
目錄
中文摘要………………………………………………………………Ι
英文摘要………………………………………………………………Ⅲ
英文縮寫表……………………………………………………………Ⅴ
誌謝……………………………………………………………………Ⅵ
第一章 文獻回顧………………………………………………………1
2.1. 肝細胞癌……………………………………………………………1
2.2. 癌症幹細胞…………………………………………………………2
2.3. 血管新生(neovascularization) ……………………………………3
2.4. 趨化因子與其接受器………………………………………………6
第二章 研究目的………………………………………………………9
第三章 研究設計………………………………………………………10
第四章 材料與方法……………………………………………………14
4.1. 儀器…………………………………………………………………14
4.2. 材料…………………………………………………………………14
4.3. 方法…………………………………………………………………18
4.3.1細胞培養……………………………………………………………18
4.3.2.pCAG-CXCR4-LacZ質體之建構………………………………………19
4.3.3.細胞X-gal染色………………………………………………………23
4.3.4.細胞之質體轉染……………………………………………………23
4.3.5.細胞之病毒感染……………………………………………………24
4.3.6.球體形成分析………………………………………………………27
4.3.7.血管形成分析………………………………………………………28
4.3.8.細胞侵襲能力分析…………………………………………………28
4.3.9.聚落形成能力分析…………………………………………………29
4.3.10.西方墨點法………………………………………………………29
4.3.11.反轉錄聚合酶鍊式反應…………………………………………33
第五章 結果……………………………………………………………37
5.1.建立抑制CXCR4表現之肝細胞癌細胞株…………………………37
5.2.建立過度表現CXCR4之肝細胞癌細胞株…………………………38
5.3.抑制CXCR4表現對肝細胞癌細胞自我新生能力之影響…………39
5.4.抑制CXCR4表現對肝細胞癌細胞增生能力之影響………………40
5.5.抑制CXCR4表現對肝細胞癌細胞侵襲能力之影響………………40
5.6.抑制CXCR4表現對肝細胞癌細胞腫瘤增生能力之影響…………41
5.7.抑制CXCR4表現對肝細胞癌細胞形成類血管能力之影響………42
5.8.過度表現CXCR4對肝細胞癌細胞自我新生能力之影響…………42
5.9.過度表現CXCR4對肝細胞癌細胞增生能力之影響………………43
5.10.過度表現CXCR4對肝細胞癌細胞侵襲能力之影響………………44
5.11.過度表現CXCR4對肝細胞癌細胞腫瘤增生能力之影響…………44
5.12.CXCR4對肝細胞癌細胞血管生成擬態相關分子之影響…………45
第六章 討論 …………………………………………………………47
第七章 結論……………………………………………………………50
第八章 參考資料………………………………………………………51
第九章 圖………………………………………………………………56
第十章 表………………………………………………………………68
第十一章 附錄………………………………………………………69

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