臺灣博碩士論文加值系統

CCN1是CCN蛋白家族中的一員，這個家族主是由一群分泌性及富含半胱胺酸(cysteine)的細胞基質蛋白所組成。CCN1參與了許多細胞生理過程，包括血管新生、胚胎發育、傷口修復及組織重建，已知CCN1失去正常的調控，與許多癌症相關。目前CCN1被認為會抑制非小細胞肺癌(NSCLCs)的生長，臨床研究顯示CCN1表現量與肺癌惡性程度呈現負相關，並且腫瘤組織高度表現CCN1時，肺癌病患的存活率增加；而TGF和肺癌的進展有很大的關聯，可藉由誘發表皮－間質轉換(EMT)協助腫瘤的進展。CCN1蛋白由四個區塊(domain)所組成，每個區塊極有可能扮演不同的的功能角色，本研究中，我們想要找出CCN1可以抑制TGF所引起的促癌活性的功能性區塊(functional domain)。
我們使用人類非小細胞肺癌細胞株A549及H520以及小鼠的肺癌細胞株LLC-1，來研究CCN1的功能，並且採用慢病毒表現系統大量表達CCN1、不同位點突變CCN1以及CCN1的蛋白質片段來探討CCN1的功能性區塊。我們發現的結果顯示TGF會誘發細胞產生趨向間質細胞形態的改變及促使細胞移動能力的增加，而CCN1可以減緩TGF1所促進的形態改變及細胞移動，因此具有抑制腫瘤的效果。而在表現CCN1第四個區塊缺陷的細胞，則無法產生抑制TGF1作用。我們的結果顯示CCN1抑制TGF的活性，主要是透過CCN1的第四個區塊(CCN1-D4)。我們進一步地檢測不同CCN1蛋白質片段對於血管新生的作用，結果顯示全長CCN1可以產生促進血管新生活性，然而CCN1-D4蛋白片段則不會引發血管新生的作用。
總結本論文，CCN1-D4可以達到與CCN1相同抑制肺癌細胞移動性的作用，值得進一步探討其是否未來可發展作為治療肺癌的蛋白質試劑。

The CCN1 protein, a member of the matricellular CCN family, is a secreted cysteine-rich matrix molecule. CCN1 regulates multiple cellular actions. Abnormal expression of CCN1 is highly associated with a variety of human diseases including multiple cancers. Studies showed that CCN1 may play a suppressing role in human non-small cell lung carcinomas (NSCLCs) that expression of CCN1 is inversely correlated with malignancy of NSCLCs and high expression levels of CCN1 associate with enhancement of patient survival. The molecular mechanism underlying CCN1-induced lung cancer suppression remains to be explored. The TGF1 is capable of inducing epithelial-mesenchymal transition (EMT) to accelerate tumor metastasis. CCN family is composed of four conserved structural domains. In the present study, we focus on structure-function relationship of the CCN1 and aim to define the structural region of CCN1 for anti-cancer activities.
We generated various CCN1 structural mutants as well as the deletion mutants that were overexpressed in lung cancer cells by the lentivirus expression strategy. Our results showed that TGF-induced EMT process and cellular migration were attenuated by the presence of CCN1 overexpression, while these effect were not observed in the cells that overexpressed CCN1 variants with defective mutations in the fourth domain. Thus, the intrinsic activity of CCN1 in suppressing NSCLC can be defined in the domain IV fragment of CCN1, the CCN1-D4. Furthermore, by in vivo angiogenesis assay, unlike the full-length CCN1, the CCN1-D4 protein was not capable of inducing angiogenesis. Taken together, the data suggest that CCN1-D4 protein fragment may be the developed as a basis of designing an effective reagent targeting NSCLCs.

致謝. i
中文摘要 iii
Abstract iv
目次 ………………………………………………………………………………………………………………………..v
圖次 ………………………………………………………………………………………………………………………vii
壹、 緒論 (Introduction) 1
一、 CCN家族 (CCN Family) 1
二、 CCN1/Cyr61 2
三、 CCN1 與癌症上的研究 3
四、 肺癌 (Lung cancer) 4
五、 表皮－間質轉換(EMT，epithelial–mesenchymal transition) 5
六、 轉化生長因子(Transforming growth factor，TGF) 6
貳、 研究動機與目的 8
參、 實驗材料與方法 (Materials and Methods) 9
一、 實驗材料（Materials） 9
(一) 細菌培養 (Bacteria Culture) 9
(二) 細胞培養 (Cell Culture) 9
(三) 基因選殖 (Molecular Cloning) 11
(四) 常用化學試劑與緩衝溶液 (Reagents and Buffer) 13
(五) 試劑套組 (Kits) 16
(六) 儀器設備 17
二、 實驗方法 (Methods) 18
(一) 細菌培養 (Bacteria Culture) 18
(二) 細胞培養 (Cell Culture) 19
(三) 基因選殖 (Molecular Cloning) 23
(四) 蛋白質相關實驗 35
(五) 抗體純化 44
(六) 人類慢病毒pLKO_AS3w.puro-CCN1的產製，以及建立內生性CCN1 被大量表現的非小細胞肺癌細胞株 47
(七) 探討CCN1調控非小細胞肺癌之相關實驗 49
(八) 探討CCN1的血管新生作用之活性於小鼠的活體實驗 55
肆、 實驗結果 (Results) 58
一、 大量表現內生性CCN1對於非小細胞肺癌細胞株的影響 58
二、 探討CCN1對TGF 誘導的EMT所扮演的角色 58
三、 利用單株抗體YM-1B對CCN1進行功能性抑制在非小細胞肺癌中 59
四、 CCN1對於TGF誘導非小細胞肺癌細胞株移動能力的影響 60
五、 大量表現內生性CCN1對於老鼠肺癌細胞株LLC-1的影響 60
六、 大量表現內生性mCCN1對於老鼠肺癌細胞株LLC-1的影響 61
七、 利用LLC-1表現CCN1進行in vivo實驗，以及觀察重組CCN1蛋白的血管新生能力 61
伍、 實驗討論(Discussion) 63
一、 探討CCN1對非小細胞肺癌扮演的角色 63
二、 探討CCN1對TGF誘導的EMT所扮演的角色 64
三、 探討CCN1抑制非小細胞肺癌移動性的功能性區塊 65
四、 mCCN1表現於老鼠肺癌細胞LLC-1抑制TGF誘導的EMT所扮演的角色 65
五、 探討CCN1對於血管新生的作用 66
六、 總結 67
陸、 參考文獻(Reference) 68
柒、 實驗圖表 (Figures and Tables) 75
捌、 附圖 (Supplementary Figures) 93
玖、 附錄 (Appendix) 101

圖一、 Lentivirus 感染非小細胞肺癌強迫內生性表現 77
圖二、 CCN1對TGF 引起非小細胞肺癌細胞株外觀上變化的影響，並抑制TGF 引起的Stress fibers 80
圖三、觀察單株抗體YM-1B對CCN進行功能性抑制後細胞型態於TGF加入後所產生的改變。 83
圖四、 單株抗體YM-1B影響CCN1抑制TGF所引起的細胞移動 85
圖五、 CCN1對TGF 引起老鼠肺癌細胞株LLC-1外觀上變化的影響，並抑制TGF 引起的EMT 88
圖六、 表現mCCN1在老鼠肺癌細胞LLC-1觀察TGF 引起的外觀上變化 90
圖七、 利用LLC-1表現CCN1進行in vivo實驗，觀察細胞成長 91
圖八、觀察重組CCN1蛋白的血管新生能力，進行in vivo實驗 92
附圖一、重組蛋白CCN1對TGF1引起外觀上變化的影響，並抑制TGF1引起的Stress fibers 94
附圖 二、YM-1E辨識各突變CCN1蛋白，而YM-1B主要辨識於CT domain的DM區域 95
附圖三、CTD-Fc及CCN1對TGF1引起外觀上變化的影響，並抑制TGF1引起的Stress fibers 97
附圖四、CTD-Fc及CCN1抑制TGF1引起EMT marker的改變 99
附圖五、CCN1抑制TGF1所引起的LLC-1細胞移動 100

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