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系統識別號 U0026-0812200910425420
論文名稱(中文) 探討MSP/RON路徑在膀胱癌細胞癌化過程的影響
論文名稱(英文) The involvement of macrophage stimulating protein (MSP)/RON pathway in the progression of human bladder cancer
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 2
出版年 92
研究生(中文) 林彥儒
研究生(英文) Yan-Ju Lin
學號 s4690401
學位類別 碩士
語文別 中文
論文頁數 88頁
口試委員 指導教授-劉校生
指導教授-周楠華
口試委員-陳鴻震
口試委員-賴明德
中文關鍵字 膀胱癌 
英文關鍵字 MSP  c-met  RTK  RON 
學科別分類
中文摘要 RON基因所控管的蛋白質產物,是具有酪胺酸酵素活性的生長因子接受體,被歸類為一種原致癌基因(proto-oncogene)。其配合基 (ligand)為巨噬細胞刺激蛋白質(macrophage stimulating protein, MSP)。MSP/RON之間的交互作用會影響細胞的各種生物現象,包括細胞分化、細胞移動、型態改變以及細胞增生。這些作用在癌症的癌化過程中,可以扮演一種促進癌化的角色。本研究主要是想了解MSP/RON相關之傳導路徑,以及在膀胱癌的癌化過程中所扮演角色。從臨床膀胱癌檢體的統計分析,發現在侵犯性膀胱癌檢體以及上泌尿道移行上皮癌的檢體中,RON之表現和膀胱癌的惡化因子有顯著的相關性,另外有2.8%的檢體顯示出RON蛋白異常分佈在細胞質中的現象。顯見RON對於膀胱癌的發生,的確扮演重要的角色,很值得進一步的釐清。
本研究使用十株不同細胞株,其中有一株正常的膀胱上皮細胞株,以及九株不同癌化等級的膀胱癌細胞株。比較RON蛋白表現及mRNA表現的分析,我們發現RON被表現在許多的腫瘤細胞中(包括RT4, TSGH8301, TCCSUP, UB37),其中以UB37的表現量最高。另外在UB40及UB47則有分子量異常的RON蛋白質表現。其中TSGH8301在MSP刺激後RON會產生大量磷酸化的現象。而UB37細胞似乎能透過自體分泌(autocrine)MSP和RON產生交互作用。除此之外,本研究也在八個臨床膀胱癌病患的尿液檢體中以及UB37的細胞培養液中發現MSP蛋白的存在,這樣的結果顯示在膀胱癌癌化過程中RON和MSP之間的關係可能是以自體分泌或是旁分泌(paracrine)的方式存在。為了進一步了解RON活化之後,所影響的生物現象。我們構築一個可受四環素調控表現的RON質體且能夠成功的在UB09細胞株中表現。結果發現RON在大量表現的情況下,的確會使細胞的生長速率、移動速率及抗細胞凋亡(anti-apoptosis)能力增強。另外以PI3K的抑制藥物wortamanin抑制PI3K活性結果發現RON所造成的細胞凋亡抑制的現象消失了。之後以報導基因(reporter gene) 研究RON可能的下游傳導路徑,結果發現當RON活化時,可能會透過PI3K-Akt活化NFkB,是否是這樣的路徑造成上述生物現象的改變,仍然須要進一步的釐清。
針對UB47細胞株,透過cDNA序列分析,在transmembrane domain前近N端處,發現有147bp核酸被刪除的現象,證實UB47所表現的RON確實為一種突變型的蛋白,另外我們也發現這個突變型的蛋白會異常的累積在細胞質中且不需MSP刺激即會產生大量磷酸化的現象。我們也利用SSCP方式分析RON的kinase domain,結果我們並沒有發現點突變的情形。我們在細胞株上所發現的現象可以解釋臨床上我們所看到的異常的RON蛋白累積的現象。另外我們也意外的發現RON蛋白的入核現象,且似乎不受MSP刺激的控制。這樣的結果或許意謂著RON入核之後也可能扮演著特殊的角色,不過這個假說尚須更深入的研究才能證實。
總結,在本研究中我們證實了RON和膀胱癌臨床發生的相關性。MSP/RON路徑在膀胱癌中可能會透過自體分泌及旁分泌的機制來進行作用。此外RON基因表現時的確會加強癌細胞的各種生物現象。而我們也發現PI3K-Akt路徑的活化和膀胱癌細胞抗凋亡的能力有關,且當其活化之後NFkB也跟著被活化,是否這樣的路徑調控了RON 所影響的生物現象則必須進一步的釐清。除此之外突變型的RON蛋白及RON入核的現象也是首次在膀胱癌中被探討。透過了解膀胱癌中MSP/RON機制,期望能夠對未來癌症的治療上有所助益。
英文摘要 RON is a transmembrane receptor tyrosine kinase encoded by RON proto-oncogene. The specific ligand of RON is macrophage-stimulating protein (MSP). The interaction of MSP and RON mediates a number of biological properties, including cell morphology, motility and proliferation. The major goal of this study is to reveal the possible MSP/RON signaling pathways in the progression of bladder carcinogenesis. By using immunostaing, we initially analyzed the correlation between RON expression and biological indicators of human bladder cancer. We found a 42.9 % and 33% expression rates for invasive bladder cancer and upper urinary tract transitional cell carcinoma, respectively. Expression of RON showed a significant association with non-papillary growth and multiple tumors of bladder cancer. In addtion, aberrant localization of RON protein in the cytosol of cancer cells was detected in about 3% of both bladder and upper tract cancer samples. Therefore, multifunctional mechanisms for RON activation seem to be involved in the progression of bladder cancer.
A total of nine bladder cancer and one immortalized uroepithelial cell line were employed in this study. Expression of wild-typed RON protein in many cancer cell lines (such as RT4, TSGH8301, TCCSUP, and UB37) were detected; while aberrant pattern of RON protein was observed in UB40 and UB47 cell lines. TSGH8301 cells showed a MSP-dependent phosphorylation of RON; but UB37 cells exhibited an autocrine MSP/RON interaction. Besides, MSP protein could be detected in the urine of bladder cancer patients (n = 8), as well as in the supernatant of UB37 cells. Taken together, both autocrine and paracrine of MSP/RON signaling appears to exist in human bladder cancer. As for biological effects of RON activation, an inducible construct of full-length RON cDNA was successfully expressed in UB09 cells. Over- expression of RON was proven to enhance the cell growth, migration, and anti-apoptosis. By using PI3K specific inhibitor (wortmannin), we confirmed that PI3K-Akt pathway involves in the RON-related anti-apoptosis. The transient reporter gene assay also confirmed the activation of NFkB by PI3K-Akt pathway.
UB47 cells were proven to have a 147bp deletion at the extracelluar domain near transmembrane region as described in the gastric cancer. The aberrant RON protein in UB47 cells accumulated in the cytoplasm, and exhibited a MSP-independent phosphorylation. But, there was no evidence of mutation in the kinase domain screened by SSCP. The results essentially support the clinical relevance of aberrant RON expression in human bladder cancer. Interestingly, we also observed a MSP-independent nuclear localization of RON protein, but the underlying mechanism and the clinical significance remain to be clarified.
Taken together, we support the clinical importance of RON in the progression of bladder carcinogenesis and the effect of MSP/RON may act through autocrine or paracrine pathway. Moreover, mutant RON is first discovered in the bladder cancer and both wild type and mutant RON could translocate into nucleus which has nerver been discussed before. We also confirmed that PI3K-Akt pathway and NFκB activation involve in the RON-related biological response, but the underlying mechanism and the clinical significance remain to be clarified. The understandings may provide a molecular basis in developing gene-targeting therapy for human bladder cancer.
論文目次 中文摘要 ..............................................................1
英文摘要 ..............................................................3
致謝 ..............................................................5
目錄 ..............................................................6
圖表目錄 ..............................................................8
縮寫及符號.............................................................10
一、緒論
I. 膀胱癌致病機轉....................................................12
II. 酪胺酸磷酸激酶14
III. MSP/RON 對於膀胱癌細胞的影響....................................15
IV. RNAi的原理及應用.................................................17
二、材料和方法
I. 菌種與細胞株......................................................19
II. 細菌培養及細胞培養...............................................19
III. 質體及其製備....................................................21
IV. 膀胱癌細胞株之RNA製備............................................23
V. 反轉錄聚合酵素連鎖反應............................................24
VI. 蛋白質電泳及西方墨...............................................27
VII. 免疫沈澱法......................................................31
VIII. RON下游報導基因分析............................................32
IX. Acridine Orange染色..............................................33
X. Time lapse recording..............................................34
XI. Subcellular Fraction of proteins extraction......................35
XII. 免疫螢光染色36
XIII. 電穿孔(electroporation)........................................37
XIV. 單股多型性聚合酶連鎖反應(SSCP)..................................38
三、結果
I. 探討RON在膀胱癌中所扮演的角色.....................................40
1. RON表現和膀胱癌發生之關係.......................................40
2. 膀胱癌細胞株RON蛋白質表現.......................................40
3. 巨噬細胞刺激蛋白質(macrophage stimulating protein, MSP)
和RON之間的交互關係..............................................41
a. 自體分泌(autocrine)及旁分泌(paracrine)........................41
b. 不同程度的RON表現特性經MSP刺激後的磷酸化影響41
II. 建立可誘導RON表現之膀胱癌細胞短暫分析模式42
III. RON基因表現後對細胞生物現象之影響分析...........................43
1. RON基因過量表現會增加細胞生長速率...............................43
2. RON過量表現使膀胱癌細胞產生抗細胞淍亡的能力.....................43
3. RON過量表現造成細胞移動特性增加.................................43
4. RON透過PI3K-Akt活化NFκB.........................................44
IV. 突變型RON蛋白功能分析及RON入核現象之探討.........................45
1. 異常分子量之RON及transmembrane domain的基因刪除..................45
2. 突變型RON在細胞質中累積的現象及RON入核特性之分析.................45
V. RON RNA interference..............................................46
四、討論...............................................................48
五、參考文獻...........................................................54
六、圖表...............................................................63
自述...................................................................88
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