請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28568完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林榮耀 | |
| dc.contributor.author | Shang-Min Lin | en |
| dc.contributor.author | 林尚民 | zh_TW |
| dc.date.accessioned | 2021-06-13T00:12:29Z | - |
| dc.date.available | 2012-08-08 | |
| dc.date.copyright | 2007-08-08 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-27 | |
| dc.identifier.citation | 1. Drucker BJ. Renal cell carcinoma: current status and future prospects. Cancer treatment reviews 2005;31(7):536-45.
2. Pavlovich CP, Schmidt LS. Searching for the hereditary causes of renal-cell carcinoma. Nature reviews 2004;4(5):381-93. 3. Lam JS, Leppert JT, Figlin RA, Belldegrun AS. Role of molecular markers in the diagnosis and therapy of renal cell carcinoma. Urology 2005;66(5 Suppl):1-9. 4. Bruno JJ, 2nd, Snyder ME, Motzer RJ, Russo P. Renal cell carcinoma local recurrences: impact of surgical treatment and concomitant metastasis on survival. BJU international 2006;97(5):933-8. 5. Jones J, Otu H, Spentzos D, et al. Gene signatures of progression and metastasis in renal cell cancer. Clin Cancer Res 2005;11(16):5730-9. 6. Hoshi S, Satoh M, Ohyama C, et al. Active chemotherapy for bone metastasis in sarcomatoid renal cell carcinoma. International journal of clinical oncology / Japan Society of Clinical Oncology 2003;8(2):113-7. 7. Tang SW, Chang WH, Chao YW, et al. Identification of differentially expressed genes in clear cell renal cell carcinoma by analysis of full-length enriched cDNA library. J Biomed Sci 2006:1-8. 8. Newton JP, Buckley CD, Jones EY, Simmons DL. Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites of PECAM-1/CD31. The Journal of biological chemistry 1997;272(33):20555-63. 9. DeLisser HM, Chilkotowsky J, Yan HC, Daise ML, Buck CA, Albelda SM. Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties. The Journal of cell biology 1994;124(1-2):195-203. 10. Muller WA, Berman ME, Newman PJ, DeLisser HM, Albelda SM. A heterophilic adhesion mechanism for platelet/endothelial cell adhesion molecule 1 (CD31). The Journal of experimental medicine 1992;175(5):1401-4. 11. DeLisser HM, Yan HC, Newman PJ, Muller WA, Buck CA, Albelda SM. Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans. The Journal of biological chemistry 1993;268(21):16037-46. 12. Prager E, Sunder-Plassmann R, Hansmann C, et al. Interaction of CD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses. The Journal of experimental medicine 1996;184(1):41-50. 13. Wong CW, Wiedle G, Ballestrem C, et al. PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis. Molecular biology of the cell 2000;11(9):3109-21. 14. Buckley CD, Doyonnas R, Newton JP, et al. Identification of alpha v beta 3 as a heterotypic ligand for CD31/PECAM-1. Journal of cell science 1996;109 ( Pt 2):437-45. 15. Chiba R, Nakagawa N, Kurasawa K, Tanaka Y, Saito Y, Iwamoto I. Ligation of CD31 (PECAM-1) on endothelial cells increases adhesive function of alphavbeta3 integrin and enhances beta1 integrin-mediated adhesion of eosinophils to endothelial cells. Blood 1999;94(4):1319-29. 16. Bird IN, Taylor V, Newton JP, et al. Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration. Journal of cell science 1999;112 ( Pt 12):1989-97. 17. Thompson RD, Wakelin MW, Larbi KY, et al. Divergent effects of platelet-endothelial cell adhesion molecule-1 and beta 3 integrin blockade on leukocyte transmigration in vivo. J Immunol 2000;165(1):426-34. 18. Newman PJ. Switched at birth: a new family for PECAM-1. The Journal of clinical investigation 1999;103(1):5-9. 19. Vivier E, Daeron M. Immunoreceptor tyrosine-based inhibition motifs. Immunology today 1997;18(6):286-91. 20. Cao MY, Huber M, Beauchemin N, Famiglietti J, Albelda SM, Veillette A. Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases. The Journal of biological chemistry 1998;273(25):15765-72. 21. Lu TT, Barreuther M, Davis S, Madri JA. Platelet endothelial cell adhesion molecule-1 is phosphorylatable by c-Src, binds Src-Src homology 2 domain, and exhibits immunoreceptor tyrosine-based activation motif-like properties. The Journal of biological chemistry 1997;272(22):14442-6. 22. Henshall TL, Jones KL, Wilkinson R, Jackson DE. Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling. J Immunol 2001;166(5):3098-106. 23. Hua CT, Gamble JR, Vadas MA, Jackson DE. Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates. The Journal of biological chemistry 1998;273(43):28332-40. 24. Pumphrey NJ, Taylor V, Freeman S, et al. Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31. FEBS letters 1999;450(1-2):77-83. 25. Pellegatta F, Chierchia SL, Zocchi MR. Functional association of platelet endothelial cell adhesion molecule-1 and phosphoinositide 3-kinase in human neutrophils. The Journal of biological chemistry 1998;273(43):27768-71. 26. Ilan N, Cheung L, Pinter E, Madri JA. Platelet-endothelial cell adhesion molecule-1 (CD31), a scaffolding molecule for selected catenin family members whose binding is mediated by different tyrosine and serine/threonine phosphorylation. The Journal of biological chemistry 2000;275(28):21435-43. 27. Vaporciyan AA, DeLisser HM, Yan HC, et al. Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo. Science (New York, NY 1993;262(5139):1580-2. 28. Bogen S, Pak J, Garifallou M, Deng X, Muller WA. Monoclonal antibody to murine PECAM-1 (CD31) blocks acute inflammation in vivo. The Journal of experimental medicine 1994;179(3):1059-64. 29. Graesser D, Solowiej A, Bruckner M, et al. Altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in PECAM-1-deficient mice. The Journal of clinical investigation 2002;109(3):383-92. 30. Brown S, Heinisch I, Ross E, Shaw K, Buckley CD, Savill J. Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature 2002;418(6894):200-3. 31. Ilan N, Mohsenin A, Cheung L, Madri JA. PECAM-1 shedding during apoptosis generates a membrane-anchored truncated molecule with unique signaling characteristics. Faseb J 2001;15(2):362-72. 32. Baldwin HS, Shen HM, Yan HC, et al. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development. Development (Cambridge, England) 1994;120(9):2539-53. 33. DeLisser HM, Christofidou-Solomidou M, Strieter RM, et al. Involvement of endothelial PECAM-1/CD31 in angiogenesis. The American journal of pathology 1997;151(3):671-7. 34. Yunmbam MK. Inhibition of breast cancer in nude mouse model by anti-angiogenesis. Oncology reports 1998;5(6):1431-7. 35. Zhou Z, Christofidou-Solomidou M, Garlanda C, DeLisser HM. Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice. Angiogenesis 1999;3(2):181-8. 36. Cao G, O'Brien CD, Zhou Z, et al. Involvement of human PECAM-1 in angiogenesis and in vitro endothelial cell migration. American journal of physiology 2002;282(5):C1181-90. 37. O'Brien CD, Cao G, Makrigiannakis A, DeLisser HM. Role of immunoreceptor tyrosine-based inhibitory motifs of PECAM-1 in PECAM-1-dependent cell migration. Am J Physiol Cell Physiol 2004;287(4):C1103-13. 38. Newman PJ, Berndt MC, Gorski J, et al. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science (New York, NY 1990;247(4947):1219-22. 39. Albelda SM, Muller WA, Buck CA, Newman PJ. Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule. The Journal of cell biology 1991;114(5):1059-68. 40. Tang DG, Chen YQ, Newman PJ, et al. Identification of PECAM-1 in solid tumor cells and its potential involvement in tumor cell adhesion to endothelium. The Journal of biological chemistry 1993;268(30):22883-94. 41. Zocchi MR, Poggi A. PECAM-1, apoptosis and CD34+ precursors. Leukemia & lymphoma 2004;45(11):2205-13. 42. Gao C, Sun W, Christofidou-Solomidou M, et al. PECAM-1 functions as a specific and potent inhibitor of mitochondrial-dependent apoptosis. Blood 2003;102(1):169-79. 43. Schneider BL, Kulesz-Martin M. Destructive cycles: the role of genomic instability and adaptation in carcinogenesis. Carcinogenesis 2004;25(11):2033-44. 44. Tanaka S, Diffley JFX. Deregulated G1-cyclin expression induces genomic instability by preventing efficient pre-RC formation. Genes Dev 2002;16(20):2639-49. 45. Ylva Hedberg EDBLGRGL. Cyclin E and p27 protein content in human renal cell carcinoma: Clinical outcome and associations with cyclin D. International Journal of Cancer 2002;102(6):601-7. 46. Hedberg Y, Roos G, Ljungberg B, Landberg G. Cyclin D3 protein content in human renal cell carcinoma in relation to cyclin D1 and clinico-pathological parameters. Acta oncologica (Stockholm, Sweden) 2002;41(2):175-81. 47. Risau W. Mechanisms of angiogenesis. Nature 1997;386(6626):671-4. 48. Pralhad T, Madhusudan S, Rajendrakumar K. Concept, mechanisms and therapeutics of angiogenesis in cancer and other diseases. The Journal of pharmacy and pharmacology 2003;55(8):1045-53. 49. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407(6801):249-57. 50. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocrine reviews 2004;25(4):581-611. 51. Schneider BP, Miller KD. Angiogenesis of breast cancer. J Clin Oncol 2005;23(8):1782-90. 52. Herbst RS, Onn A, Sandler A. Angiogenesis and lung cancer: prognostic and therapeutic implications. J Clin Oncol 2005;23(14):3243-56. 53. Kabbinavar F, Hurwitz HI, Fehrenbacher L, et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003;21(1):60-5. 54. Rmali KA, Puntis MC, Jiang WG. Tumour-associated angiogenesis in human colorectal cancer. Colorectal Dis 2007;9(1):3-14. 55. Kosharskyy B, Solban N, Chang SK, Rizvi I, Chang Y, Hasan T. A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer. Cancer research 2006;66(22):10953-8. 56. Baldewijns MM, Thijssen VL, Van den Eynden GG, et al. High-grade clear cell renal cell carcinoma has a higher angiogenic activity than low-grade renal cell carcinoma based on histomorphological quantification and qRT-PCR mRNA expression profile. British journal of cancer 2007;96(12):1888-95. 57. Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal and pathological cell motility. Int J Biochem Cell Biol 2004;36(10):1890-909. 58. Distler JH, Hirth A, Kurowska-Stolarska M, Gay RE, Gay S, Distler O. Angiogenic and angiostatic factors in the molecular control of angiogenesis. Q J Nucl Med 2003;47(3):149-61. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28568 | - |
| dc.description.abstract | 腎細胞癌是由腎小管上皮細胞病變所衍化而成的癌症，佔所有的惡性腫瘤中的百分之二，也是最致命的一種腎臟癌類。目前最常見的腎細胞癌類型是透明細胞型，而大部分的腎細胞癌對目前，因此目前為止手術根除是最主要的治療方式。然而五年的存活率仍低於百分之二十，除此之外臨床上還沒有可作為預測診斷或治療預後的分子指標。因此，我們希望藉由研究腎細胞癌相關基因來了解腎細胞癌形成的分子機制。在先前的研究中，本實驗室的唐賽文先生建構了腎細胞癌以及正常腎臟組織的全長cDNA庫，希望藉由比較兩者之間不同基因的表現來找出與腎細胞癌形成的相關基因。經過比較之後，發現血小板內皮細胞黏附因子-1 f 這個基因在腎細胞癌中的表現有上升的情形。
PECAM-1 是一種細胞附著分子，一開始被發現於在血液中循環的血小板及白血球細胞表面之上，其主要功能是參與細胞和細胞間的連結。近年來發現PECAM-1除了原本的功能之外，還能作為訊息傳遞中轉接分子 (adaptor molecules) 的一個支架(scaffold) ，參與細胞內訊息傳遞。最近的研究指出PECAM-1在血管新生的過程中，可能扮演一個重要的角色，然而PECAM-1在血管新生及細胞癌化的過程中，所參與的完整分子機制至今還不是非常的清楚。 在本篇研究中我們發現了PECAM-1 的mRNA含量在臨床採集的腎細胞癌組織中有表現量上升的情形。之後我們在轉染PECAM-1腎細胞癌的細胞株 (HEK293)中，利用了共軛焦顯微鏡觀察到PECAM-1在細胞中主要存在在細胞膜上。而在功能性的研究中，我們發現在HEK293細胞中過度表現PECAM-1會造成調控細胞週期G1/S時期的細胞週期素D1(CCND1)基因的mRNA及蛋白表現量的上升，同時細胞株轉殖PECAM-1時其生長速率也較快。除此之外我們還觀察到血管新生因子(VEGF-A)，會因為PECAM-1的過度表現而造成mRNA及蛋白含量的上昇，其細胞的移動速率增快也在傷口癒合測試中 (Wound healing assay) 及細胞穿透測試 (transwell assay) 中可觀察得到。綜合以上的結果，我們認為在腎細胞癌中過度表現的PECAM-1不但可以使得細胞比較有生長的優勢，同時也使得細胞處於較癌化的形態。 | zh_TW |
| dc.description.abstract | Renal cell carcinoma (RCC) was thought to be one of major types of human kidney cancers, and it is characterized by a high mortality rate and a high resistance to chemotherapeutic treatment as well as radiations. The surgery remains to be the main method of treatment. Thus, we try to identify the genes associated with RCC to investigate the molecular mechanism of RCC. In previous studies, full-length enriched cDNA libraries of ccRCC and normal kidney tissues were constructed by Mr. Sai-Wen Tang from this laboratory. By comparing the differential gene-expression profiles of ccRCC and normal tissues, the platelet endothelial cell adhesion molecule (PECAM-1) was found to be up-reglulated significantly in ccRCC tissues.
PECAM-1 is a cell adhesion molecule expressed on the surface of circulating platelets and leukocytes, and it was also shown to be a scaffold for signaling and adaptor molecules for signal transduction in cells. It was demonstrated recently that PECAM-1 is associated in angiogenesis, but the molecular mechanism remained unclear.These findings suggest that PECAM-1 might play a crucial role in tumor development and progression. In present study, the expression of PECAM-1 gene was found upregulated in mRNA level of ccRCC tissue pairs. Confocal microscopy studies showed that PECAM-1 localized in plasma membrane. By functional studies, we have demonstrated that overexpression of PECAM-1 in HEK293 cell line induced the upregulation of a G1/S cell cycle regulatory gene, CCND1, and it also enhances the proliferation rate of transfected cells. Furthermore, overexpression of PECAM-1 also upregulated mRNA and protein levels of anginogenesis factor VEGF-A and further promoted the migration ability of HEK293 cells. Taken together, the results suggested that overexpression of PECAM-1 in ccRCC could provide several cell growth advantages and tumorgenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T00:12:29Z (GMT). No. of bitstreams: 1 ntu-96-R94442011-1.pdf: 1123309 bytes, checksum: f926b845b52cfc5cf856fcb92664923c (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | Index
摘要 i Abstract iii Index v Introduction 1 Materials and methods 6 1. Materials 6 2. Quantitative Real-time RT PCR (Q-PCR) 7 3. Plasmid construction 8 3.1 Preparation of Luria-Bertani (LB) medium and plates 8 3.2 PCR amplifaication 8 3.3 Direct cloning by TOPO○R system 9 3.4 Transformation 10 3.5 Screening of the transformants by colony PCR 10 3.6 Plasmid minipreparation 11 3.7 Restriction enzyme digestion 11 4. Cell culture 12 5. Transient transfection 12 5.1 Generating PECAM-1 stable cell lines 12 6. Western blotting 13 6.1 Preparation of cell lysates and condition medium 13 6.2 Quantification of protein by bicinchoninic (BCA) assay 13 6.3 Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 14 6.3.1 Preparation of SDS-polyacrylamide gels 14 6.3.2 Preparation of protein samples and polyacrylamide gel electrophoresis 15 6.4 Transferring the protein using a Semi-Dry System 16 6.5 Immunoblotting 17 7. RNAs extraction and reverse transcription 17 7.1 Extraction of total RNAs from transfected cells 17 7.2 Treatment of DNA 18 7.3 Reverse transcription 19 8. Immunofluorescence and confocal microscopy 19 8.1 Transfection and fixation 19 8.2 Permeabilization and blocking 19 8.3 Immunostaining 19 9. Cell Migration Assay 20 9.1 Transwell cell motility assay 20 10. MTT assay 21 11. Flow cytometric analysis of DNA content 22 11.1 Ethanol fixation 22 11.2 DNA staining 22 Results 24 1. PECAM-1 mRNA level was upregulated in ccRCC tissues 24 2. Cloning of PECAM-1 into the expression vector 25 3. PECAM-1 expressed in transient transfected and PECAM-1 stable expressed HEK293 cells. 25 4. Overexpression of PECAM-1 up-regulates cell cycle regulators CCND1. 26 5. Overexpression of PECAM-1 up-regulates anginogenesis gene , VEGF-A. 27 6. Overexpression PECAM-1 enhanced cell migration 28 Discussion 29 Legends 33 Fig.1 Up-regulation of PECAM-1 mRNA in forty ccRCC tissue pairs 33 Fig.2 Restriction enzymes digestion and agarose gel electrophoresis of pcDNA-PECAM-1 construct. 33 Fig.3 PECAM-1 was expressed ectopically in HEK293 cells. 34 Fig.4 Intracellular localization of PECAM-1 in HEK293 cell line 34 Fig.5 PECAM-1 was sustained expressed in HEK293 cell. 34 Fig.6 Overexpression of PECAM-1 accelerates the proliferation of HEK293 cells 35 Fig.7 Overexpression of PECAM-1 up-regulates CCND1 expression 35 Fig.8 Overexpression of PECAM-1 increased the S phase of HEK293-PECAM-1 stable cell lines as measured by flow cytometric analysis. 36 Fig.9 Overexpression of PECAM-1 up-regulates VEGF-A expression. 36 Fig.10 Analysis of overexpression of PECAM-1 induced cell motility measured by wound healing assay. 37 Fig.11 Analysis of the PECAM-1 overexpression promoted cell motility by Using transwell cell motility assay. 37 Figures 38 References 49 Table of Figures Fig.1 Up-regulation of PECAM-1 mRNA in forty ccRCC tissue pairs 38 Fig.2 Restriction enzymes digestion and agarose gel electrophoresis of pcDNA-PECAM-1 construct. 39 Fig.3 PECAM-1 was expressed ectopically in HEK293 cells. 40 Fig.4 Intracellular localization of PECAM-1 in HEK293 cell line 41 Fig.5 PECAM-1 was sustained expressed in HEK293 cell. 42 Fig.6 Overexpression of PECAM-1 accelerates the proliferation of HEK293 cells 43 Fig.7 Overexpression of PECAM-1 upregulates CCND1 expression 44 Fig.8 Overexpression of PECAM-1 increased the S phase of HEK293-PECAM-1 stable cell lines as measured by flow cytometric analysis. 45 Fig.9 Overexpression of PECAM-1 up-regulates VEGF-A expression. 46 Fig.10 Analysis of overexpression of PECAM-1 induced cell motility measured by wound healing assay. 47 Fig.11 Analysis of the PECAM-1 overexpression promoted cell motility by Using transwell cell motility assay. 48 | |
| dc.language.iso | en | |
| dc.title | 透明細胞型腎細胞癌相關基因:血小板內皮細胞黏附分子I(PECAM-1)之功能性研究 | zh_TW |
| dc.title | Studies on the Function of ccRCC-Associated Gene Platelet Endothelial Cell Adhesion Molecule I (PECAM-1) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 周玉山,吳華林,李明學 | |
| dc.subject.keyword | 腎細胞癌,血小板內皮細胞黏附分子I,細胞週期素D1,血管新生因子A, | zh_TW |
| dc.subject.keyword | ccRCC,PECAM-1,VEGF-A,CCND1, | en |
| dc.relation.page | 52 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-28 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-96-1.pdf 目前未授權公開取用 | 1.1 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。