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研究生:林涵澤
研究生(外文):Lin,Han Tze
論文名稱:探討在前列腺癌轉移中matriptase和N-acetylglucosamnyltransferaseV基因所扮演之功能
論文名稱(外文):To Evulate the Function of Matriptase and N-acetylglucosamnyltransferase V in Prostate Cancer Metastasis
指導教授:莊宏亨
指導教授(外文):Juang, Horng Heng
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:69
中文關鍵詞:前列腺癌前列腺
外文關鍵詞:matriptase
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在前列腺癌中,其轉移之機制到目前還不是很明瞭。從高轉移能力的前列腺癌細胞, PC-3分離出一株細胞, PC-J可以用來篩選可能的轉移基因。藉由short tandem repeat 分析證明PC-3與PC-J為同源的前列腺癌細胞。分別把PC-3及PC-J細胞注射至裸鼠前列腺,8個星期後顯示注射PC-3細胞的組別有較小的腫瘤但是有轉移的現象;而注射PC-J細胞的組別有較大的腫瘤但沒有發現轉移的現象。在細胞生長及侵入試驗中都顯示這兩株細胞都有不同的特性。另外從微陣列分析方法、RT-PCR及西方點墨法都說明matriptase在前列腺癌可能是一個與轉移有關的基因。同時在酵素分析中指出LNCaP細胞,有較低的轉移能力,但是有很高的matriptase基因的表達。將使用RT-PCR和lectin blot等實驗方法來更進一步的研究在前列腺癌中表達的基因 N-acetylglucosaminyltansferase V (MGAT5) ;將MGAT5於LNCaP細胞中過度表現可以明顯的增加matriptase的活性。同時於PC-3細胞中過度表達bikunin基因會抑制matriptase的表現。另外利用RNAi的方法剔除PC-3細胞的MGAT5基因其侵入的能力明顯的被抑制下來。由這些結果指出matriptase和MGAT5基因可能在前列腺癌的轉移中扮演著重要的角色。
The mechanisms of metastasis in prostatic tumor are still unknown. A subclone cell line (PC-J) is isolated from a high metastasis human prostate cell line, (PC-3), to screen a putative metastatic gene. PC-3 and PC-J cells are identified as homologous cells by short tandem repeat analysis. When cells are prostatic-injection to the nude mice, respectively, for 8 weeks, results showed PC-3 injected-group has low tumor volume but presents a phenomenon of metastasis, whereas in PC-J injected-group presents high tumor volume without metastasis. In vitro cell proliferation assay and invasion assay revealed the different characteristics of tumorigenesis and invasion between PC-3 and PC-J cells. Results from microarray assay, RT-PCR and immunoblot assay suggested that matriptase is a putative metastatic gene in human prostate. The zymography assay indicated that LNCaP cells, a low metastatic prostatic tumor cells, express high level of matriptase but have extreme low activity of matriptase. Further studies using RT-PCR and lectin blot assay indicated that N-acetylglucosaminyltransferase V (MGAT5) is constituently expressed in prostate carcinoma cells; however, transient overexpression of MGAT5 significantly enhances activity of matriptase in LNCaP cells. When bikunin was overexpressioned into PC-3 cells which blocked the expression of matriptase or when MGAT5 was knock-down from PC-3 cells, the invasion ability of PC-3 cells was dramatic decrease by using in vitro invasion assay. Our studies suggest that matriptase and MGAT5 may play important roles in the metastasis of prostate cancer.
授權書.......................................................................................................iii
誌謝…………………………………………………………………...…iv
中文摘要....................................................................................................v
英文摘要...................................................................................................vi
目錄..........................................................................................................vii
圖目錄.......................................................................................................ix
I.前言..........................................................................................................1
1.前列腺癌...............................................................................................1
2.Matriptase基因的功能.......................................................................4
3.低氧與腫瘤生長關係.........................................................................6
4.N-acetylglucosamnyltransferase V (MGAT5)基因的功能................9
5.bikunin基因的功能............................................................................10
II.材料與方法..........................................................................................13
1.Cell Culture and Cell Condition.........................................................13
2.Animal Model.....................................................................................13
3.RNA Extraction…………………………………………………..14
4.Reverse Transcription for Synthesis of cDNA……………………15
5.Polymerase Chain Reaction (PCR)………………………………..16
6.Western Blot………………………………………………………17
7.Gelatin Zymography………………………………………………19
8.Luciferase Assay…………………………………………………..19
9.Plasmid Construction……………………………………………...21
10.Construction of Overexpression Plasmid………………………..22
11.Cell Proliferation Assay………………………………………….23
12.Wound Healing Assay…………………………………………...23
13.Cell Invasion Assay……………………………………………...23
14.Lectin Blot……………………………………………………….24
15.Stable Clone Establish…………………………………………...25
16.RNA Interference…………………………………………………..26
III.結果……...………………………………………………………...27
1.PSA基因於前列腺癌細胞之表現……………………………………..27
2.從PC-3分離出一株子細胞, PC-J……………………………………...27
3.PSA基因啟動子/強化子於LNCaP、PC-3及PC-J之表達………….28
4. PC-3與PC-J細胞注射於裸鼠前列腺中之生長情形………………...28
5.利用RT-PCR、西方點墨法來觀察PC-3與PC-J細胞間之差異…...29
6.觀察st14與MGAT5基因之活性於LNCaP、PC-3和PC-J細胞…..30
7.觀察LNCaP、PC-3與PC-J細胞侵入之能力……………………….….31
8.將MGAT5基因過度表達於LNCaP細胞…………………………..…31
9.將bikunin基因過度表現於PC-3細胞……………………………..….31
10.剔除MGAT5基因於PC-3細胞………………………………………32
11.低氧對st14基因啟動子的調控………………………………………33
IV.討論…...………………………………………………………………...34
V.圖表…………………………………………………………………..36
VI.文獻………………………………………………………………...61
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