跳到主要內容

臺灣博碩士論文加值系統

(3.236.110.106) 您好!臺灣時間:2021/07/24 06:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:李和俊
研究生(外文):He-Chun Li
論文名稱:前列腺癌之蛋白質表現分析
論文名稱(外文):Protein expression analysis of prostate cancer
指導教授:黃弘文黃弘文引用關係
指導教授(外文):Hurng-Wern Huang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:62
中文關鍵詞:基質輔助雷射脫附游離飛行時間質譜二維電泳反轉錄聚合酶連鎖反應良性前列腺增生癌症前列腺
外文關鍵詞:2 dimensional electrophoresisANXA3ProstateMALDI-TOFSPIN2MAP2K5cancerBenign Prostatic Hyperplasia2-DEKRT8MayvenPGAM1ACPPBPHRT-PCRTAGLN2
相關次數:
  • 被引用被引用:0
  • 點閱點閱:206
  • 評分評分:
  • 下載下載:39
  • 收藏至我的研究室書目清單書目收藏:0
前列腺癌是西方國家男性中最常見的癌症之一種,但是如果能早期發現,存活率非常高,前列腺特異性抗原(prostate specific antigen, PSA)是目前最常用來檢測前列腺癌的血清腫瘤標誌,但PSA的使用上仍然不夠方便,因為無法完全分辨前列腺癌與良性前列腺腫大之病人,在近年來的研究中,有許多有潛力腫瘤標誌仍在研究中,例如: free prostate specific antigen(f-PSA), complexed prostate specific antigen (C-PSA)和kallikrein等。綜合我們之前的實驗發現,Mayven蛋白質在癌症病人血清中及組織中表現量增加,並利用RT-PCR確認Mayven基因的表現,在良性前列腺增生(benign prostate hyperplasia,BPH)組織中Mayven的mRNA表現量,在三個BPH組織的檢體中,分別是正常組織的5.0,6.9及11.3倍,而在癌症組織中Mayven的mRNA表現量,以TNM分期,在T1中為正常組織的12.7倍,在T1a中為正常組織的4.7倍,在T2a中為正常組織的0.8倍,在T2b中平均後為正常組織的2.3倍,在T3a中為正常組織的0.5倍,在T3b中為正常組織的0.1倍;結果顯示Mayven在良性前列腺增生的組織中及T1a分期以前的早期癌症組織中,與正常組織的表現量相比,皆在4.76倍之上;而在T2a分期之後皆在3.7倍以下,表示Mayven的表現隨著癌化過程中,具有表現量下降的現象。在本實驗中利用二維電泳(two-dimensional electrophoresis)與基質輔助雷射脫附游離飛行時間(matrix- assisted laser desorption ionization-time of flight, MALDI-TOF)質譜分析,發現7個在癌症與正常組織中具差異表現的蛋白質,經MS-Fit工具比對出為Keratin 8(KRT8), MAPK kinase 5(MAP2K5), Acid phosphatase (ACPP), Annexin A3(ANXA3), Phosphoglycerate mutase 1 (PGAM1), Spindlin-like protein 2 (SPIN2) and Transgelin 2(TAGLN2)。
Prostate cancer is one of the most common malignant tumors in solid organs of old men. However, the patients are nearly 100 percent survivable if detected early. Prostate-specific antigen (PSA) is a valuable prostate cancer biomarker that is now wildly used for population screening, diagnosis, and monitoring of patients with prostate cancer. But PSA is not good enough for a biomarker because it can not distinguish benign prostate hyperplasia (BPH) from prostate tumor . Recently, there are some tumor marker still in study, for example: free prostate specific antigen(fPSA), complexed prostate specific antigen (C-PSA), kallikrein,prostate specific membrane antigen (PSMA). From previously study,we had found many different protein expressions between serum of normal and prostate patients. Mayven is one of the novel proteins that had been identified. The mRNA expression of Mayven in prostate cancer tissue is determined by quantitative RT-PCR. The result shows that the mRNA expression of Mayven in Benign Prostatic Hyperplasia (BPH) is about 5.0-11.3 fold than normal tissue , 12.7 fold in Prostate Cancer (PCa) stage T1 and 0.1- 3.7 fold after cancer stage T2. The Mayven gene expression is predominate in tumor stage T1, decrease after T2 stage. However the expressed pattern of mayven in BPH remains further investigation due to the limited sample size. Furthermore, with 2 dimensional electrophoresis (2–DE), we have found 7 differentially expressed proteins between tissue of normal and prostate patients, and these proteins are identified by MALDI-TOF mass spectrometry and MS-Fit. These identified proteins are Keratin 8(KRT8), MAPKkinase5(MAP2K5), Acid phosphatase (ACPP), Annexin A3(ANXA3), Phosphoglycerate mutase 1 (PGAM1), Spindlin-like protein 2 (SPIN2) and Transgelin 2(TAGLN2).
致謝辭
中文摘要
英文摘要
壹、 序言 頁數
1. 前列腺介紹與前列腺癌-----------------------------------1
2. 前列腺炎與良性前列腺腫大-------------------------------2
3. 前列腺的診斷及治療方式---------------------------------2
4. 前列腺癌的分期-----------------------------------------4
5. Mayven基因的介紹---------------------------------------5
6. 實驗目標-----------------------------------------------5
貳、實驗材料和方法
1 檢體來源-----------------------------------------------6
2 定量反轉錄聚合酶鍊鎖反應(Quantitative RT-PCR)
2.1 primer(引子) 設計-------------------------------6
2.2 全核糖核酸(total RNA)萃取-----------------------6
2.3 mRNA反轉錄成cDNA-------------------------------7
2.4 即時PCR反應的操作和定量分析--------------------7
3 蛋白質二維電泳
3.1 血清之處理--------------------------------------8
3.2 組織蛋白質萃取----------------------------------8
3.3 蛋白質定量--------------------------------------8
3.4 Isoelectric focusing electrophoresis (IEF)---------------9
3.5 DryStrip equilibration------------------------------10
3.6 Sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)-------------------------------------10
3.7 Coomassie brilliant blue staining ---------------------11
3.8 Silver staining ------------------------------------11
3.9 Gel imaging analysis -------------------------------11
3.10 In gel digestion-----------------------------------12
3.11 MALDI-TOF質譜操作----------------------------13
3.12 使用MS-Fit辨認未知蛋白質-----------------------13
肆、實驗結果-----------------------------------------------14
伍、討論---------------------------------------------------17
陸、參考文獻-----------------------------------------------21
柒、表-----------------------------------------------------29
捌、圖-----------------------------------------------------36
玖、附錄---------------------------------------------------47
Bander,N.H. MHC class I and II expression in prostate carcinoma and modulation by interferon-alpha and -gamma. Prostate 33, 233-239 (1997).
Bangma,C.H. & Verhagen,P.C. Blood and serum substances for markers of prostate cancer. Microsc. Res. Tech. 51, 430-435 (2000).
Bosland,M.C., Prinsen,M.K., Rivenson,A. & Weisburger,J.H. Induction of skin and thyroid tumors in male rats by N-methyl-N- nitrosourea after sequential treatment with cyproterone acetate and testosterone propionate: effects of castration, rat strain and time of carcinogen injection. Carcinogenesis 13, 669-674 (1992).
Bruchovsky,N. & Wilson,J.D. The conversion of testosterone to 5-alpha-androstan-17-beta-ol-3-one by rat prostate in vivo and in vitro. J. Biol. Chem. Carter,B.S. Allelic loss of chromosomes 16q and 10q in human prostate cancer. Proc. Natl. Acad. Sci. U. S. A 87, 8751-8755 (1990).
Casanova, M. L.; Bravo, A.; Ramirez, A.; Morreale de Escobar, G.; Were, F.; Merlino, G.; Vidal, M.; Jorcano, J. L. Exocrine pancreatic disorders in transsgenic (sic) mice expressing human keratin 8. J. Clin. Invest. 103: 1587-1595, (1999).
Caskey,C.T., Pizzuti,A., Fu,Y.H., Fenwick,R.G. Jr. & Nelson,D.L. Triplet repeat mutations in human disease. Science 256, 784-789 (1992).
Catalona,W.J. Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective
multicenter clinical trial. JAMA 279, 1542-1547 (1998).
Choe,L.H. & Lee,K.H. A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell. Electrophoresis 21, 993-1000 (2000).
Christensson,A., Laurell,C.B. & Lilja,H. Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur. J. Biochem. 194, 755-763 (1990).
Dhanasekaran,S.M. Delineation of prognostic biomarkers in prostate cancer. Nature 412, 822-826 (2001).
Elghany,N.A., Schumacher,M.C., Slattery,M.L., West,D.W. & Lee,J.S. Occupation, cadmium exposure, and prostate cancer. Epidemiology 1, 107-115 (1990).
Espana,F. Quantitative immunoassay for complexes of prostate-specific antigen with alpha2-macroglobulin. Clin. Chem. 42, 545-550 (1996).
Gao,X., Honn,K.V., Grignon,D., Sakr,W. & Chen,Y.Q. Frequent loss of expression and loss of heterozygosity of the putative tumor suppressor gene DCC in prostatic carcinomas. Cancer Res. 53, 2723-2727 (1993) Gerner,C. Elevated plasma levels of crosslinked fibrinogen gamma-chain dimmer indicate cancer-related fibrin deposition and fibrinolysis. Thromb. Haemost. 85, 494-501 (2001).
Gianazza,E. & Arnaud,P. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA. Biochem. J. 201 , 129-136 (1982).
Giovannucci,E. The CAG repeat within the androgen receptor gene and its relationship to prostate cancer. Proc. Natl. Acad. Sci. U. S. A 94, 3320-3323 (1997).
Giroldi,L.A. & Schalken,J.A. Decreased expression of the intercellular adhesion molecule E-cadherin in prostate cancer: biological significance and clinical implications. Cancer Metastasis Rev. 12, 29-37 (1993).
Gumerlock,P.H., Poonamallee,U.R., Meyers,F.J. & deVere White,R.W. Activated ras alleles in human carcinoma of the prostate are rare. Cancer Res. 51, 1632-1637 (1991).
Hachulla,E., Laine,A. & Hayem,A. Alpha 1-antichymotrypsin microheterogeneity in crossed immunoaffinoelectrophoresis with free concanavalin A: a useful diagnostic tool in inflammatory syndrome. Clin. Chem. 34, 911-915 (1988).
Hara,M., Inoue,T., Koyanagi,Y., Yamazaki,H. & Fukuyama,T. [Immunoelectrophoretic studies of the protein components in human seminal plasma (especially its specific component). (Forensic immunological study of body fluids and secretions. VI)]. Nippon Hoigaku Zasshi 23, 117-122 (1969).
He, T.; Stepulak, A.; Holmstrom, T. H.; Omary, M. B.; Eriksson, J. E. The intermediate filament protein kinase 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase. J. Biol. Chem. 277: 10767-10774,(2002).
Ittmann,M.M. Chromosome 10 alterations in prostate adenocarcinoma (review). Oncol. Rep. 5, 1329-1335 (1998).
Konig,J.J. Cytogenetic characterization of several androgen responsive and unresponsive sublines of the human prostatic carcinoma cell line LNCaP. Urol. Res. 17, 79-86 (1989).
Konishi,N. K-ras activation and ras p21 expression in latent prostatic carcinoma in Japanese men. Cancer 69, 2293-2299 (1992).
Kunimi,K., Bergerheim,U.S., Larsson,I.L., Ekman,P. & Collins,V.P. Allelotyping of human prostatic adenocarcinoma. Genomics 11, 530-536 (1991).
Lara,P.N., Jr. & Meyers,F.J. Treatment options in androgen-independent prostate cancer. Cancer Invest 17, 137-144 (1999).
Lauber,W.M.. Mass spectrometry compatibility of two-dimensional gel protein stains. Electrophoresis 22, 906-918 (2001).
Lilja,H. & Abrahamsson,P.A. Three predominant proteins secreted by the human prostate gland. Prostate 12, 29-38 (1988).
Lilja,H. A kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein. J. Clin. Invest 76, 1899-1903 (1985).
Lollo,B.A.. Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClear. Electrophoresis 20, 854-859 (1999).
Lovgren,J., Rajakoski,K., Karp,M., Lundwall a & Lilja,H. Activation of the zymogen form of prostate-specific antigen by human glandular kallikrein 2. Biochem. Biophys. Res. Commun. 238, 549-555 (1997).
Lundgren,R.. Cytogenetic analysis of 57 primary prostatic adenocarcinomas. Genes Chromosomes. Cancer 4, 16-24 (1992).
Luo,J. alpha-Methylacyl-CoA Racemase: A New Molecular Marker for Prostate Cancer. Cancer Res. 62, 2220-2226 (2002).
Mack,P.C. Increased RB1 abnormalities in human primary prostate cancer following combined androgen blockade. Prostate 34, 145-151 (1998).
McCombs,R.M. Role of oncornaviruses in carcinoma of the prostate. Cancer Treat. Rep. 61, 131-132 (1977).
Mehta PB, Jenkins BL, McCarthy L, Thilak L, Robson CN, Neal DE, Leung HY.MEK5 overexpression is associated with metastatic prostate cancer, and stimulates proliferation, MMP-9 expression and invasion.Oncogene. Mar 6;22(9):1381-9(2003).
Meyers,F.J. Very frequent p53 mutations in metastatic prostate carcinoma and in matched primary tumors. Cancer 83, 2534-2539 (1998).
Miettinen M, Fetsch JF.Distribution of keratins in normal endothelial cells and a spectrum of vascular tumors: implications in tumor diagnosis. Hum Pathol. Sep;31(9):1009-10(2000).
Mills,P.K., Beeson,W.L., Phillips,R.L. & Fraser,G.E. Dietary habits and breast cancer incidence among Seventh-day Adventists. Cancer 64, 582-590 (1989).
Mulloy R, Salinas S, Philips A, Hipskind RA., Activation of cyclin D1 expression by the ERK5 cascade. Oncogene. Aug 21;22(35):5387-98(2003).
Pylkkanen,L., Makela,S. & Santti,R. Animal models for the preneoplastic lesions of the prostate. Eur. Urol. 30, 243-248 (1996).
Oh B, Hwang SY, Solter D, Knowles BB .Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo. Development. Jan;124(2):493-503(1997).
Qian,Y. Two-dimensional gel electrophoresis detects prostate-specific antigen- alpha1-antichymotrypsin complex in serum but not in prostatic fluid. Clin. Chem. 43, 352-359 (1997).
Reed,J.C. Regulation of apoptosis by bcl-2 family proteins and its role in cancer and chemoresistance. Curr. Opin. Oncol. 7, 541-546 (1995).
Rooney,C., Beral,V., Maconochie,N., Fraser,P. & Davies,G. Case-control study of prostatic cancer in employees of the United Kingdom Atomic Energy Authority. BMJ 307, 1391-1397 (1993).
Roger S Kirby, Timothy J Christmas, Michael K Brawer. Prostate Cancer nd.Mosby.London.(2001).
Ryu JW, Kim HJ, Lee YS, Myong NH, Hwang CH, Lee GS, Yom HC.The proteomics approach to find biomarkers in gastric cancer. J Korean Med Sci. Aug;18(4):505-9(2003).
Sinha,P., Poland,J., Schnolzer,M. & Rabilloud,T. A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two- dimensional electrophoresis. Proteomics. 1, 835-840 (2001).
Soltysik-Espanola,M. Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain. Mol. Biol. Cell 10, 2361-2375 (1999).
Stamey,T.A. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N. Engl. J. Med. 317, 909-916 (1987).
Steele,R., Lees,R.E., Kraus,A.S. & Rao,C. Sexual factors in the epidemiology of cancer of the prostate. J. Chronic. Dis. 24, 29-37 (1971).
Sumner,L.W., Wolf-Sumner,B., White,S.P. & Asirvatham,V.S. Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 16, 160-168 (2002).
Sumner,L.W., Wolf-Sumner,B., White,S.P. & Asirvatham,V.S. Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 16, 160-168 (2002).
Takahashi,S. Frequent loss of heterozygosity at 7q31.1 in primary prostate cancer is associated with tumor aggressiveness and progression. Cancer Res. 55, 4114-4119 (1995).
Takayama,T.K., Fujikawa,K. & Davie,E.W. Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein. J. Biol. Chem. 272, 21582-21588 (1997).
Vlietstra,R.J., van Alewijk,D.C., Hermans,K.G., van Steenbrugge,G.J. & Trapman,J. Frequent inactivation of PTEN in prostate cancer cell lines and xenografts. Cancer Res. 58, 2720-2723 (1998).
Wang,M.C. Prostate antigen: a new potential marker for prostatic cancer. Prostate 2, 89-96 (1981).
Whitesel J,Donohue R,MAni J .Acid phosphatase:Its influence on the management of carcinoma of the prostate.J Urol 1984;131:70-72.
Zhang J, Yu L, Fu Q, Gao J, Xie Y, Chen J, Zhang P, Liu Q, Zhao S.Mouse phosphoglycerate mutase M and B isozymes: cDNA cloning, enzyme activity assay and mapping. Gene. Feb 21;264(2):273-9(2001).
Zhau,H.E. Biomarkers associated with prostate cancer progression. J. Cell Biochem. Suppl 19, 208-216 (1994).
Zhau,H.E., Wan,D.S., Zhou,J., Miller,G.J. & von Eschenbach,A.C. Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines. Mol Carcinog. 5, 320-327 (1992).
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top