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研究生:曹經漢
研究生(外文):Ching-Han Tsao
論文名稱:於大腸直腸癌中找出與癌症轉移相關的基因
論文名稱(外文):Identification of a metastasis related gene in colon cancer
指導教授:黃智生黃智生引用關係
指導教授(外文):Jason C. Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:52
中文關鍵詞:結腸直腸癌癌症轉移脂肪分化相關蛋白
外文關鍵詞:colorectal cancermetastasisADFP
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結腸直腸癌(CRC)在台灣以及全世界都是一種常見的惡性腫瘤。大約有將近有半數患有結腸直腸癌的患者可以藉由手術與抗癌藥物的合併治療達到治癒的目的,但是癌症轉的轉移往往會造成治療失敗。為了研究與癌症轉移有關的基因的研究,我們使用微陣列分析的方法分析不同時期結腸直腸癌患者的基因表現,藉由比對原發性腫瘤與轉移性腫瘤之間基因表現的差異,我們一共得到262個可能與癌症轉移相關的基因。再利用RT-PCR與real-time PCR去驗證262個當中還沒有文獻報導與結腸直腸癌轉移有直接關係的11個基因,透過此一方式我們將有可能的候選者縮小到四個,分別是ADFP、SDC4、ENPEP和ANLN。我們首先先將注意力集中於ADFP這個基因上,截至目前為止有關於ADFP這個基因的功能仍然不太清楚。根據前人的研究發現這個基因參與在脂肪細胞的分化,但是它與癌症之間的關係仍有待研究。當使用real-time PCR分析不同時期臨床病人的檢體,我們發現到ADFP基因的表現會隨者臨床時期的增加而變多,這個結果顯示ADFP的表現可能會因為癌症的生成與轉移而有所增加。為了去證明這項假設,我們將ADFP這個基因大量表現於合適的結腸直腸癌細胞中進行日後的實驗。首先我們利用real-time PCR的方式檢測四株結腸直腸癌細胞株SW480、SW620、HCT-15和HT-29內生性ADFP mRNA的表現,發現SW480在內生性的ADFP有最低的表現,所以此株細胞株適合做為ADFP基因大量表現。此外,我們也發現由於ADFP的半生期相當的短暫,所以最佳的表現時間為轉染後24至36小時之間。結合上述結果,我們利用wound healing和細胞侵襲分析去證明當ADFP在結腸直腸癌細胞中大量表現時是否會增強癌細胞的移動力和侵襲能力。我們發現當使用ADFP表現質體去轉染癌細胞時,在48小時後,這些細胞將culture insert所造成的間隙填滿的速度較快。而在細胞侵襲分析方面,同樣我們也發現到在細胞轉染36小時後,與vector control相比的確會有較多的細胞穿過matrigel爬到transwell的底面。結合上述兩項實驗的結果,我們可以證明的確當ADFP在結腸直腸癌細胞中大量表現時,可以增進癌細胞的移動力和侵襲力,但是我們仍需要更多的臨床證據去支持我們的假設,就是ADFP基因的大量表現是促進結腸直腸癌的轉移的原因之ㄧ。
Colorectal cancer(CRC)is one of the most common malignant neoplasms in Taiwan and the world. About half of patients with colorectal cancer can be cured by surgery and multimodal treatment. But metastasis is the most important cause for cure failure. We used microarray to analyze the gene expression profiles in colorectal cancer specimens. By comparing primary and metastatic carcinomas, we obtained 262 metastasis related genes. RT-PCR and real-time PCR were used to verify 11 of the 262 genes. We further narrowed down to four possible candidates:ADFP, SDC4, ENPEP and ANLN. First, we focused on adipose differential related protein (ADFP). So far, the function of ADFP is not clear. According to previous studies, it was involved in adipocyte differentiation, but its relationship with cancer is unknown. Based on real-time PCR result, we found that the expression level of ADFP is higher in patients’biopsies of more advanced tumor grade. This implied that the amounts of ADFP are increased during cancer progression and metastasis. In order to prove the hypothesis, we expressed ADFP in suitable colon cancer cell line. For the purpose, we utilized qPCR to quantify endogenous ADFP expression level in SW480, SW620, HCT-15 and HT-29. SW480 has lower endogenous mRNA expression level of ADFP than the others, so SW480 is an appropriate cell line for overexpressing ADFP. Besides, we also optimize the time point for ADFP expression. Our results showed that the maximum expression of ADFP could be detected 24 to 36 hours post-transfection. Combining the two results, we used wound healing and invasion assay to validate whether ADFP overexpression will increase in vitro cell mobility and invasive ability of cancer cells. We used wound healing to verify that cancer cells mobility was enhanced in SW480 overexpressing ADFP. In wound healing assay, we found that the gap was filled up faster with SW480 transfected with ADFP expression plasmid compared with vector control plasmid in 48 hours. In invasion assay, when ADFP was overexpressed in SW480, the number of migrating cells crossing coated matrigel was more than that of vector control. Taken together, our findings shows that ADFP can enhance in vitro cells mobility and invasive ability of colon cancer cells, but more evidence will be needed to support our assumption that the ADFP gene expression can promote tumor metastasis.
中文摘要 1
Abstract 3
第一章 緒論 5
第一節 結腸直腸癌(Colorectal cancer;CRC) 5
第二節 脂肪分化相關蛋白(adipose differentiation-related     protein;ADFP)簡介 7
第三節 癌症的轉移(Metastasis) 8
第四節 研究動機與目的 8
第二章 材料與方法 10
第一節 實驗材料 10
1. 勝任細胞株(Competent cell strain) 10
2. 細胞株(Cell lines) 10
3. 質體(Plasmids) 11
4. 限制酶及酵素酵素(Restriction endonuclease and enzymes) 11
5. 抗體(Antibodies) 11
6. 實驗套組(Kits) 12
7. 細胞培養液(Culture medium) 12
8. 緩衝溶液(Buffer) 13
9. DNA電泳之用 14
10. 蛋白質電泳之用 14
11. 西方墨點轉漬檢視之用 15
12. 勝任細胞製備之用 16
第二節 實驗方法 18
1. 總核醣核酸萃取(Total RNA extraction from cell line) 18
2. 反轉錄聚合酶鏈鎖反應(Reverse transcription polymerase   chain reaction;RT-PCR) 18
3. 聚合酶鏈鎖反應(Polymerase chain reaction;PCR) 19
4. 利用即時聚合酶鏈鎖反應比較目標基因在不同時期病人臨床檢體 的表現反轉錄聚合酶鏈鎖反應(Reverse transcription polymerase chain reaction;RT-PCR) 20
5. 即時聚合酶鏈鎖反應(Real-time polymerase chain reaction; RT-PCR) 20
6. ADFP基因表現質體的建構 20
7. 小量質體DNA之萃取(Mini plasmid DNA extraction) 23
8. 中量質體DNA之萃取(Midi plasmid DNA extraction) 24
9. 酒精沉澱(Ethanol precipitation) 25

10. 短暫性轉染作用(Transient transfection) 25
11. 慢病毒之製備(Lentivirus production) 26
12. 慢病毒之感染(Lentivirus infection) 27
13. 西方墨點法(Western blot) 27
14. 細胞移動分析(Migration assay) 29
15. 細胞侵襲分析(Invasion assay) 30
第三章 實驗結果 31
1. 利用RT-PCR和Real-time PCR在結腸直腸癌細胞株中驗證微陣列分 析所得到的基因 31
2. 藉由Real-time PCR分析不同時期臨床病患的檢體來驗證微陣列分 析的結果 32
3. 構築ADFP大量表現之質體 32
4. 利用Real-time PCR與Western blot找出最適合表現ADFP之細胞株 與時間點 33
5. 利用wound healing進行細胞移動力的分析 34
6. 利用細胞侵襲分析研究細胞的侵襲能力 34
第四章 實驗討論 35
第五章 參考文獻 39
第六章 實驗結果圖表 42
1.Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer   statistics, 2009. CA Cancer J Clin 2009;59(4):225-49.
2.Cappell MS. Pathophysiology, clinical presentation, and   management of colon cancer. Gastroenterol Clin North Am   2008;37(1):1-24, v.
3.Heald RJ, Bussey HJ. Clinical experiences at St. Mark's   Hospital with multiple synchronous cancers of the colon   and rectum. Dis Colon Rectum 1975;18(1):6-10.
4.Cappell MS. From colonic polyps to colon cancer:      pathophysiology, clinical presentation, and diagnosis.   Clin Lab Med 2005;25(1):135-77.
5.HJR B. Familial polyposis coli: family studies,       histopathology, differential diagnosis and results of    treatment. Baltimore (MD): Johns Hopkins University Press  1975.
6.Stryker SJ, Wolff BG, Culp CE, Libbe SD, Ilstrup DM,    MacCarty RL. Natural history of untreated colonic polyps.  Gastroenterology 1987;93(5):1009-13.
7.Compton CC. Colorectal carcinoma: diagnostic, prognostic,  and molecular features. Mod Pathol 2003;16(4):376-88.
8.Ricci-Vitiani L, Pagliuca A, Palio E, Zeuner A, De Maria  R. Colon cancer stem cells. Gut 2008;57(4):538-48.
9.de la Chapelle A. Genetic predisposition to colorectal   cancer. Nat Rev Cancer 2004;4(10):769-80.
10.Boland CR. Understanding familial colorectal cancer-   finding the corner pieces and filling in the center of   the puzzle. Gastroenterology 2004;127(1):334-8.
11.Veale A. Intestinal polyposis. Eugenis Laboratory     Memoirs Series 40. New York:Cambridge University Press   1965.
12.Herrera L, Kakati S, Gibas L, Pietrzak E, Sandberg AA.   Gardner syndrome in a man with an interstitial deletion   of 5q. Am J Med Genet 1986;25(3):473-6.
13.Bodmer WF, Bailey CJ, Bodmer J, et al. Localization of   the gene for familial adenomatous polyposis on chromosome  5. Nature 1987;328(6131):614-6.
14.Kinzler KW, Nilbert MC, Su LK, et al. Identification of  FAP locus genes from chromosome 5q21. Science 1991;253   (5020):661-5.
15.Peltomaki P, Aaltonen LA, Sistonen P, et al. Genetic   mapping of a locus predisposing to human colorectal     cancer. Science 1993;260(5109):810-2.
16.Fishel R, Lescoe MK, Rao MR, et al. The human mutator   gene homolog MSH2 and its association with hereditary    nonpolyposis colon cancer. Cell 1993;75(5):1027-38.
17.Bronner CE, Baker SM, Morrison PT, et al. Mutation in   the DNA mismatch repair gene homologue hMLH1 is       associated with hereditary non-polyposis colon cancer.   Nature 1994;368(6468):258-61.
18.Papadopoulos N, Nicolaides NC, Wei YF, et al. Mutation   of a mutL homolog in hereditary colon cancer. Science    1994;263(5153):1625-9.
19.Kolodner RD, Hall NR, Lipford J, et al. Structure of the  human MSH2 locus and analysis of two Muir-Torre kindreds  for msh2 mutations. Genomics 1994;24(3):516-26.
20.Ivanovich JL, Read TE, Ciske DJ, Kodner IJ, Whelan AJ. A  practical approach to familial and hereditary colorectal  cancer. Am J Med 1999;107(1):68-77.
21.Heid HW, Schnolzer M, Keenan TW. Adipocyte         differentiation-related protein is secreted into milk as  a constituent of milk lipid globule membrane. Biochem J   1996;320 ( Pt 3):1025-30.
22.Heid HW, Moll R, Schwetlick I, Rackwitz HR, Keenan TW.   Adipophilin is a specific marker of lipid accumulation in  diverse cell types and diseases. Cell Tissue Res 1998;294 (2):309-21.
23.Shaw CS, Sherlock M, Stewart PM, Wagenmakers AJ.      Adipophilin distribution and colocalization with lipid   droplets in skeletal muscle. Histochem Cell Biol 2009;131(5):575-81.
24.Saarikoski ST, Rivera SP, Hankinson O. Mitogen-inducible  gene 6 (MIG-6), adipophilin and tuftelin are inducible by  hypoxia. FEBS Lett 2002;530(1-3):186-90.
25.Marin HE, Peraza MA, Billin AN, et al. Ligand activation  of peroxisome proliferator-activated receptor beta     inhibits colon carcinogenesis. Cancer Res 2006;66(8):4394- 401.
26.Yao M, Tabuchi H, Nagashima Y, et al. Gene expression   analysis of renal carcinoma: adipose differentiation-   related protein as a potential diagnostic and prognostic  biomarker for clear-cell renal carcinoma. J Pathol     2005;205(3):377-87.
27.Steeg PS. Tumor metastasis: mechanistic insights and    clinical challenges. Nat Med 2006;12(8):895-904.
28.Thiery JP. Epithelial-mesenchymal transitions in tumour  progression. Nat Rev Cancer 2002;2(6):442-54.
29.Naldini L, Weidner KM, Vigna E, et al. Scatter factor   and hepatocyte growth factor are indistinguishable     ligands for the MET receptor. EMBO J 1991;10(10):2867-78.
30.蘇琡涵. Mechanism of metastasis in colorectal cancer. 陽 明大學微生物及免疫學研究所 碩士論文 2008.
31.Bender FC, Reymond MA, Bron C, Quest AF. Caveolin-1    levels are down-regulated in human colon tumors, and    ectopic expression of caveolin-1 in colon carcinoma cell  lines reduces cell tumorigenicity. Cancer Res 2000;60   (20):5870-8.
32.Garber K. Energy boost: the Warburg effect returns in a  new theory of cancer. J Natl Cancer Inst 2004;96(24):1805- 6.
33.Shaw RJ. Glucose metabolism and cancer. Curr Opin Cell   Biol 2006;18(6):598-608.
34.Bui T, Thompson CB. Cancer's sweet tooth. Cancer Cell   2006;9(6):419-20.
35.Garber K. Energy deregulation: licensing tumors to grow.  Science 2006;312(5777):1158-9.
36.Clemens MJ. Targets and mechanisms for the regulation of  translation in malignant transformation. Oncogene 2004;23 (18):3180-8.
37.Averous J, Proud CG. When translation meets        transformation: the mTOR story. Oncogene 2006;25(48):6423- 35.
38.Voeller D, Rahman L, Zajac-Kaye M. Elevated levels of   thymidylate synthase linked to neoplastic transformation  of mammalian cells. Cell Cycle 2004;3(8):1005-7.
39.Rahman L, Voeller D, Rahman M, et al. Thymidylate     synthase as an oncogene: a novel role for an essential   DNA synthesis enzyme. Cancer Cell 2004;5(4):341-51.
40.Kuhajda FP. Fatty-acid synthase and human cancer: new   perspectives on its role in tumor biology. Nutrition    2000;16(3):202-8.
41.Menendez JA, Lupu R. Fatty acid synthase and the      lipogenic phenotype in cancer pathogenesis. Nat Rev     Cancer 2007;7(10):763-77.
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