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標題: | 可溶性Contactin 4於大腸直腸癌細胞抑癌功能之研究 Study of tumor suppressor functions of soluble Contactin 4 in colorectal cancer |
作者: | Yi-Hsuan Hsu 許翊萱 |
指導教授: | 楊雅倩(Ya-Chien Yang) |
關鍵字: | 大腸直腸癌,Contactin 4,抑癌基因,可溶性 CNTN4,血管新生, Colorectal cancer,Contactin 4,Tumor suppressor gene,Soluble CNTN4,Angiogenesis, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 大腸直腸癌於全世界及臺灣均是癌症死亡原因的前三名,且在臺灣大腸直腸癌已攀升為每年發生率最高之癌症。於人類癌症常見抑癌基因座因基因組缺失造成抑癌基因靜默,本實驗室先前的研究,於人類第三號染色體 3p25.3-p26.3鑑定 Contactin 4 (CNTN4) 於大腸直腸癌可能扮演抑癌基因的角色。於大腸直腸癌細胞株 HCT116大量表現 CNTN4可抑制細胞增生、固著依賴性及非固著依賴性胞落形成的能力,同時,在活體動物實驗則發現表現 CNTN4 可以抑制 HCT116細胞於裸鼠皮下腫瘤的生成及減少腫瘤的血管密度,初步亦觀察到可溶性 CNTN4可抑制細胞增生之能力。目前可溶性 CNTN4 的功能尚未清楚,本論文分為三個部分探討: (一) 為探討可溶性 CNTN4 發展成為抗癌藥物的潛力,首先將分泌型 CNTN4 表現質體以轉染方式送入 HCT116 和 HEK293 細胞,使其表現可溶性 CNTN4,並以細胞模型進行功能性試驗,結果顯示含有可溶性 CNTN4 之條件培養液可抑制細胞增生、固著依賴性及非固著依賴性胞落形成之能力。(二) 為進一步瞭解 CNTN4 之抑癌作用區域,故建構兩種分泌型截斷 CNTN4 (truncated CNTN4) 表現質體:pSecTag2/sCNTN4_IGc2 和 pSecTag2/sCNTN4_FN3,以轉染方式送入HCT116 和 HEK293 細胞,確認其可正確表現,之後即可用於體外細胞功能性試驗,以探討其對大腸直腸癌細胞之抑制作用。(三) 為探討可溶性 CNTN4抑制體外血管生成試驗之形成管狀結構能力,首先建立體外血管生成試驗之實驗條件,後續即可用於可溶性全長 CNTN4 和兩種截斷 CNTN4 之研究。綜合目前結果,可溶性 CNTN4 可抑制大腸直腸癌細胞株增生、固著依賴性及非固著依賴性胞落形成之能力,相似於細胞膜之CNTN4 皆對大腸直腸癌細胞株具有生長抑制的功能。 Colorectal cancer (CRC) is one of the top three leading causes of cancer death in the world and Taiwan. Nowadays, CRC is the cancer with the highest annual incidence in Taiwan. Genomic deletion at tumor suppressor loci is common in human cancers and contributes to tumor suppressor genes (TSGs) silencing. In our previous study, we have identified Contactin 4 (CNTN4) as a novel tumor suppressor gene located at chromosome 3p25.3-p26.3 that is silenced or markedly down-regulated in CRC cell lines and colorectal carcinomas compared with their matched normal mucosae. Ectopic expression of CNTN4 indeed reduced cell proliferation, anchorage-dependent and -independent colony formation of CRC HCT116 cells in vitro. Subcutaneous injection of CNTN4-expressing CRC cells in BALB/c nude mice showed the reduction of tumor growth and less microvessel density compared with that observed in Mock-inoculated mice. In addition, the conditioned medium containing soluble CNTN4 (sCNTN4) could suppress cell growth of HCT116. The function of sCNTN4 is still not clear currently. In the study, there are three specific aims. First, to explore the potential of sCNTN4 for an innovative anti-cancer protein drug, the secretory CNTN4-expressing plasmid, pSecTag2/sCNTN4 was transiently transfected into HCT116 and HEK293 cells. By in vitro cell functional assays, we demonstrated that the conditioned media containing sCNTN4 could inhibit the cell proliferation, anchorage-dependent and -independent colony formation of HCT116 cells. As for the second aim, in terms of small molecule protein drugs, we attempted to part the soluble full-length CNTN4 into two truncated proteins containing immunoglobulin-like C2 (IGc2) domains and fibronectin type-III (FN3) domains separately. To identify whether both of truncated proteins could suppress tumor behaviors, we have constructed the soluble truncated CNTN4-expressing plasmids, pSecTag2/sCNTN4_IGc2 and pSecTag2/sCNTN4_FN3, which exhibit correct expression in HCT116 and HEK293 cells by transient transfection. In the third part, to investigate whether sCNTN4 could inhibit angiogenesis by in vitro angiogenesis assay, the experimental conditions for in vitro angiogenesis assay to test the ability of forming tubular structures have been established, followed by studies of soluble full-length CNTN4 and truncated CNTN4. Taken together, the conditioned media containing soluble CNTN4 could inhibit cell proliferation, anchorage-dependent and anchorage-independent colony formation of CRC cells in vitro. Either membrane-anchored CNTN4 or soluble CNTN4 could inhibit cell growth of CRC. Our findings illustrate CNTN4 as a novel human TSG and suggest an antitumor potential of soluble CNTN4 for a therapeutic drug. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70342 |
DOI: | 10.6342/NTU201803375 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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