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系統識別號 U0026-0812200914025446
論文名稱(中文) 探討內源性纖連蛋白與癌細胞表面上聚合體纖連蛋白組裝的關聯性
論文名稱(英文) The Involvement of Endogenous Fibronectin in Polymeric Fibronectin Assembly on Cancer Cell Surfaces
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 95
學期 2
出版年 96
研究生(中文) 沈佩蓁
研究生(英文) Pei-Jan Shen
學號 s1693404
學位類別 碩士
語文別 中文
論文頁數 97頁
口試委員 口試委員-吳華林
口試委員-高銘欽
召集委員-吳昭良
指導教授-鄭宏祺
口試委員-賴明德
中文關鍵字 癌症轉移  纖連蛋白 
英文關鍵字 metastasis  fibronectin 
學科別分類
中文摘要 近來許多研究指出,癌細胞並不是隨機地轉移至身體其他各處,癌症轉移是具有組織特異性。而造成組織特異性的轉移主要是藉由癌細胞和繼發器官(secondary organ)上內皮細胞(endothelial cell)間的結合。在實驗室之前的研究發現,大白鼠乳癌細胞(MTF7 Cells)能成功地轉移到肺臟是藉由懸浮癌細胞表面上所組裝的多聚體纖連蛋白(polymeric fibronectin;ploly-FN)跟肺臟內皮細胞表面之第四型雙肽胜肽水解酶(Diepeptidy peptidase IV;DPPIV)結合,在懸浮癌細胞經由血流流經肺臟微血管時被肺臟內皮細胞所捕捉進而造成癌細胞侵入形成繼發瘤(secondary tumor)。這些懸浮癌細胞表面上所組裝的poly-FN,其來源有:懸浮癌細胞本身所分泌的內源性纖連蛋白(endogenous fibronectin;endogenous FN)、及血液中的外源性纖連蛋白(exogenous fibronectin;exogenous FN)。有趣的是,我們發現愈惡性的懸浮癌細胞能自行合成endogenous FN的能力越強,且能在細胞表面上組裝大量的多聚體纖連蛋白(poly-FN)。因此,懸浮癌細胞能在表面上組裝多聚體纖連蛋白和懸浮癌細胞合成endogenous FN的能力存在著很強的關聯性。於是,我們推測endogenous FN可能參與調控懸浮癌細胞表面上poly-FN的組裝。本論文選用小片段核糖核酸干擾技術來探討這個可能性。但是,由實驗室經驗得知,這些由上皮細胞癌化的惡性腫瘤細胞是很難利用核苷核酸轉染(DNA transfection)的方式將小片段核糖核酸送進細胞中。所以本實驗企圖利用重組腺病毒能感染廣泛腺體性上皮細胞的能力,來解決轉染上皮細胞低效率的問題,並且我們也利用比RNA polymerase typeⅠ、typeⅡpromoter轉錄能力更強的RNA polymerase typeⅢ promoter中的modifed tRNAmet-derived (MTD) promoter來加強FN siRNA的表達。我們已經成功的獲得攜帶有小片段干擾RNA的重組腺病毒,而這些重組腺病毒都能有效的感染標的細胞:如MTF7 (大白鼠乳癌細胞)、4T1(老鼠乳癌細胞)。而我們利用收集condition media的方式已發現,所挑選的小片段干擾RNA可以有效的抑制癌細胞合成的endogenous FN。利用RT-PCR也發現小片段干擾RNA確實能辨認纖連蛋白的序列且達到分解的效果。此外利用免疫螢光染色的方式,也可以發現:送入小片段干擾RNA的懸浮癌細胞(MTF7、4T1),其細胞表現上所組裝的poly-FN有減少的現象。利用流式細胞儀定量的方式,也可以發現利用小片段干擾RNA使得endogenous FN合成量下降後,進而可以使得懸浮癌細胞組裝poly-FN的能力下降。未來,我們可以建立in vivo實驗 ,來證明這些癌細胞是否也會造成肺臟轉移的現象有下降的趨勢。一但確定endogenous FN確實參與在poly-FN組裝的過程,藉由抑制endogenous FN的形成將可以成為抑制癌症轉移的有效策略之一 。
英文摘要 There is mounting evidence showing that metastasis is an organ-specific rather than a random pattern of dissemination. Organ-specific metastasis is indispensably dictated by adhesion events occurring between tumor cells and organ-specific vascular endothelia. We have identified Dipeptidyl peptidase IV (DPP IV) expressed on rat lung capillary endothelia and polymeric Fibronectin (polyFN) assembled on suspended breast cancer cell surfaces as a receptor/ligand pair in mediating lung colonization. This cancerous polyFN assembly results from both endogenous and exogenous sources of dimeric FN. Interestingly, we found that in contrast to their non-metastatic counterparts, many lung-metastatic cancer cell lines invariably carry strong messages for endogenous FN synthesis and are able to assemble polyFN on their surfaces. We rationalize that endogenously synthsized FN may be involved in polyFN assembly on suspended cancer cell surfaces. This possibility can be explored by examining the surface polyFN assembly of suspended cancer cells whose endogenous FN is post-transcriptionally knocked-down by RNA interference (RNAi) techniques. To overcome the common difficulties of liposomal transfecting molecules into epithelial-origin cancer cells, we employed replication-deficient recombinant adenoviruses to deliver the DNA construct encoding a specific FN small interfering RNA (FN siRNA) known to effectively knock-down endogenous FN expression. We also used a modified tRNAmet-derived (MTD) promoter which was more effective in driving the expression of ribozymes than other Pol II and Pol III promoters to drive the cellular expression of FN siRNA. We have successfully isolated the well-packed viruses containing a FN short hairpin RNA (FN shRNA) sequence from HEK293 cells, and, as expected, tremendously improved the delivery of FN siRNA into the target cells, MTF7, a rat breast cancer cell line and 4T1, a mouse breast cancer cell line . We collected the serum-free condition media and demonstrated a significant decrease in endogenous FN secretion upon FN siRNA treatment. To firmly assure the knock-down effect by detecting FN mRNA level, we used techniques such as RT-PCR. Also, we found that the polyFN assembly on suspended cancer cell surfaces was decrease upon FN siRNA treatment by anti-FN Ab immunofluorescent staining and FACS. We intend to establish in vivo experiment to test polyFN down regulation upon endogenous FN inhibition will prevent lung colonization.Once the relationship between endogenous FN and polyFN is ascertained, the inhibition of suspended cancer cell polyFN assembly by down-regulating the endogenous FN could potentially be used as an anti-metastatic strategy for cancer therapy.
論文目次 中 文 摘 要 I
英 文 摘 要 Ⅲ
誌 謝 V
目 錄 Ⅵ
表目錄 Ⅹ
圖目錄 ⅩI
附圖目錄 XIII
第一章 緒 論 1
第一節 臟器特異性癌症轉移 1
1.1 癌症的轉移 1
1.2 臟器特異性轉移 2
1.3 DPPIV/FN 與臟器特異性轉移 4
第二節 纖連蛋白 4
2.1 纖連蛋白的構造 5
2.2 纖連蛋白的功能 5
2.3 癌症細胞上聚合性纖連蛋白與轉移的關聯性 5
2.4 纖連蛋白與細胞之關係 6
第三節 核糖核酸干擾現象 8
3.1 核糖核酸干擾的發現 8
3.2 核糖核酸干擾的機制 9
3.3 以載體系統表現siRNA 9
第四節 利用腺病毒來運送小片段核糖核酸 10
4.1 利用病毒載體運送外來基因進入動物細胞表現 10
4.2 腺病毒的構造 11
4.3 重組腺病毒的載體 11
4.4 重組腺病毒應用的優缺利弊 12
第五節 研究動機 12
第二章 材料與方法 14
1.DNA基本技術操作 14
1-1 抽取質體DNA 14
1-2 瓊脂膠電泳分析 16
1-3 以電泳法回收DNA 16
1-4 限制酶切割 18
1-5 接合反應 18
1-6 大腸桿菌勝任細胞製作 19
1-7 形質轉移 20
1-8 長期保存菌種 21
2.基本細胞培養技術 21
2-1 繼代細胞 21
2-2 冷凍保存細胞 24
2-3 解凍冷凍保存細胞 24
3.DNA轉染系統 24
3-1 細胞計數 25
3-2 轉染DNA(傳統法) 25
3-3 轉染DNA(使用Invitrogen lipofectamineTM-plusTM kit) 26
4. 生產腺病毒 27
4-1 於細菌中取得腺病毒表現載體 27
4-2 以Ad293或HEK293生產腺病毒 28
5.蛋白質基本技術操作: 30
5-1 以TCA濃縮蛋白 30
5-2 蛋白濃度測定 31
5-3 SDS-PAGE製備 31
5-4 SDS-PAGE電泳分析 33
5-5 西方墨點法 34
6.RT-PCR(反轉錄聚合酶鏈鎖反應) 35
6.1 抽取RNA 35
6-2 反轉錄反應(Reverse Transcription) 36
6-3 聚合酶鏈鎖反應 37
7.免疫螢光染色(Immunofluorescence staining)及FACS 38
第三章 結果 40
1.構築具有可以辨認內源性纖連蛋白的序列的shRNA 40
2.獲得攜帶有小片段干擾RNA的重組腺病毒 43
3.攜帶有小片段干擾RNA的重組腺病毒具有感染標的細胞的能力 45
4.小片段干擾RNA可以抑制內源性纖連蛋白的合成 46
5.抑制內源性纖連蛋白的合成可以使得細胞表面所組裝的多聚
體纖連蛋白減少 47
6. 結論 49
第四章 討論 50
1.內源性纖連蛋白(endogenous FN)、多聚體纖連蛋白
(poly-FN)、癌症轉移間的關聯性 50
2.利用小片段Fn siRNA knockdown內源性纖連蛋白的效率 50
3.使用腺病毒載體(adenovirus delivery)的優點與缺點 51
4.endogenous FN的表現(synthesis)與分泌(secretion)的關係 52
5.當內源性纖連蛋白合成量下降,細胞表面上組裝的多聚體纖
連蛋白也有下降的現象 53
6.利用抑制endogenous FN的方式來達到抑制轉移 54
第五章 參考文獻 55
第六章 表 67
第七章 圖 69
第八章 附圖 92
作者自述 97
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