臺灣博碩士論文加值系統

大腸直腸癌一直是許多西方工業化國家中常見的癌症之一，在許多國家中，以美國為例，近幾年來大腸直腸癌均居於所有致死癌病中的第二位，估計在美國每年有 15 萬人以上得此病症。西風東漸，西式飲食的盛行，導致台灣地區大腸直腸癌發生率有日益升高的趨勢，根據衛生署的統計，大腸直腸癌的發生率由 1980 年每十萬人口中之 5. 95 人，增加至 1990 年的 12.29 人，而至 1997年公佈的大腸直腸癌發生率，己攀升至 13.2人。
一般認為大腸直腸癌的發生與飲食、遺傳等因素均有關係，其預防之道乃在於早期發現早期施於治療，其存活率仍極高。隨著生物醫學及分子生物技術的日新月異，對大腸直腸癌癌化的分子機轉有更多的認識，但仍難以一窺全貌。雖截至目前為止己有許多與大腸直腸癌相關的基因被報告出來，如: C-myc、ras、DCC、MCC、P53等；此些因可能是產生突變或具有差異表現，但相信有更多的基因參與癌症發生的過程，根據文獻報告在正常與癌症組織之間約有百分之一的基因具有差異表現，即大約 2000 個基因左右。因此探求其他參與大腸直腸癌癌化基因,對於了解致癌的過程能有更進一步暸解，相信對於大腸直腸癌的診斷、預防及治療會有所幫助。
本論文實驗第一部份乃承先前利用差異表現法,研究分化型與未分化型甲狀腺癌細胞株，發現一具差異表現的基因- Matrix Gla protein，推測此基因亦可能在大腸直腸癌中參與癌化過程或分化程度或轉移之可能。因此，以此基因為探針利用 Northern blo t的方法進行大腸直腸癌組織與正常組織對的篩檢。結果發現在80組檢體中有76%，其癌組織之MGP 之mRNA 表現量下降，甚至不表現的情形。
另外，以cDNA Microarray此項技術研究正常組織與癌組織及原位癌的大腸直腸癌細胞株與轉移性的大腸直腸癌細胞株，基因的差異表現情形，發現在588個己知與癌症相關的基因中：在組織檢體的研究結果方面，有11個基因在癌組織有超量表現的情形，另有55個基因在正常組織在超量表現。在癌細胞株的研究中有36個基因在轉移癌細胞株(CoLo 205)有超量表現；有50個基因在原位癌細胞株(CoLo 320DM)有超量表現。

Colorectal cancer has been listed as the major cause of death among cancers for most western development nations, and as the top three in Taiwan area in 1997. If diagnosed at its early staging, the 5-year survival rates will be higher than 50%. However, the lack of an effective early-detection screening methods is the major barrier to improve the treatment of patients with colorectal cancer. For of reason of this searching for biomarkers and under-standing the more molecular biological information about colorectal cancer has become the most important issue now, since such markers will useful and helpful on early diagnosis, treatment and prognosis of this disease.
Although several genes have been reported to be mutated or differentially expressed in colorectal cancer, a much larger number of genes are likely to be involved in the transformation of colorectal epithelial cells.
In this study, Part1:we use the matrix Gla protein MGP gene as a probe to analysis the expression volume of the RNA level in colorectal cancer, in the present study we found that the MGP expression is down regulated in 75% of colorectal cancer compared with their pair normal tissue. This finding suggest that the loss of MGP expression may be associated with tumor progression and metastasis. Part 2:Using the technique of differential hybridization of AtlasTM Human Cancer cDNA expression array to study the differences in gene expression between difference biological conditions. Auto-radiographic analysis showed that of the 588 genes analyzed, 55 are over-expressed in normal colorectal tissue and 14 in Duke*s A tumor tissue. 40 are over-expressed in Duke*s B tumor tissue and 40 in Duke*s C Tumor tissue. 36 are over-expressed in CoLo 205 cell line and 48 in CoLo 320DM cell line.

第一章 緒論 1
第一節 研究目的 1
第二節 研究方法 2
第二章 文獻回顧 6
第一節 大腸直腸解剖組織學及其生理機能 6
1.1.大腸直腸解剖組織學 6
1.2.大腸直腸之生理機能 7
第二節腸直腸癌的分期 7
第三節大腸直腸癌的流行病學特徵 8
3.1.生活環境與大腸直腸癌 8
3.2.年齡與大腸直腸癌 9
3.3.發生部位與大腸直腸癌 9
3.4.性別與大腸癌 9
3.5.種族宗教與大腸直腸癌 10
第四節大腸直腸癌的致病因 11
4.1.飲食習慣 11
4.2.基因遺傳因素 12
4.3.發炎性腸疾病 12
4.4.多發性大腸息肉症 12
4.5.家族性息肉症 13
4.6.家族性大腸直腸癌病史 13
第五節大腸直腸癌的症狀 13
第六節大腸直腸癌的診斷 14
6.1.肛門指診 14
6.2.糞便潛血檢查 15
6.3.大腸X光鋇鹽造影 16
6.4.內視鏡檢查 16
6.5.組織切片 16
6.6.腫瘤標記 17
第七節大腸直腸癌的治療 18
7.1. 外科切除手術 18
7.2. 放射線療法 18
7.3. 化學治療法 19
第八節大腸直腸癌的癌基因變化 20
8.1. c-myc 基因 20
8.2. ras 基因 21
8.3. 基因對 (allele )之缺損,癌抑制基因 21
8.4. MCC,APC gene 22
8.5. P53 基因 23
8.6. DCC 基因 23
第三章材料與方法 25
第一節儀器 25
第二節材料 24
2.1.大腸直腸癌組檢體來源與取樣方法 26
2.2.細胞株 26
2.3.培養基 27
第三節方法 27
3.1.Total RNA 的抽取 27
3.2.Tota lRNA 品質分析 28
3.3.Total RNA 轉漬 29
3.4.MGP cDNA探針製備 30
3.5.結腸直腸癌細胞株之培養與收集. 35
3.6.Total RNA之萃取 35
3.7.Total RNA品質的確認 35
3.8.Poly A+ mRNA 之純化 36
3.10.MGP cDNA probes的製備 37
3.11.Column chromatography 37
3.12.Scintillation counting 38
3.13.Hybridizating cDNA probe to the AtlasTM Array 39
3.14.基因放射強度之定量 40
3.15.特異基因之 RT-PCR分析 40
3.16.差異表現基因-cdc25B基因在臨床檢體之表現 41
第四章結果與討論 43
4.1.Total RNA 的萃取及其品質分析 43
4.2.MGP PCR 再放大及限制酵素確定 MGP 片段 44
4.3.北方墨點法進行臨床大腸直腸檢體篩選 44
4.4.統計 45
4.5.探討 Poly A+ mRMA 的純化及品質再確定 46
4.6.AtlasTM Human Cancer cDNA Expression Array結果 47
4.7.特異基因之RT-PCR分析 48
4.8.cdc25B基因在臨床檢體之表現 49
第五章 結論與未來展望 51
5.1.The expression of MGP in colorectal cancer 51
5.2.AtlasTM Human Cancer cDNA Expression Array 53
參考文獻 95
附錄一 民國86年台灣地區國人 102
附錄二 結腸直腸解剖圖 103
附錄三 結腸直腸橫剖面圖 104
附錄四 MGP基因之鹼基與氨基酸序列 105
附錄五 Altas TM Human Cancer cDNA Expresion Array 實驗原理與流程圖 106
附錄六 Altas TM Human Cancer cDNA Expresion Array 基因功能分類圖 107
附錄七 結腸直腸癌分子致癌機制模式圖 108

1. Khoury, D. A., F.G. Opelka, and D.E. Beck, et al. 1996.Colon surveillance after colorectal cancer surgery. Disease of the Colon and Rectum 39(3): 252-256.
2. 行政院衛生署1998. 行政院衛生署公報.
3.J .M. Moertel, J.R." Molecular Themes in Oncogenesis.
[Review] Cell, 64, 235- 48.
4. C.G. Moertel, J.r. O*Fallon, V. Go, M.J. O*Connell, and G.S. thynne (1986). The Preparative Carcinoembryonic Antigen test in the Diagnosis, Staging and Diagnosis of Colorectal Cancer. Cancer, 58, 603-10.
5. M.J. Duffy (1995). "Can Molecular Markers Now Be Used For Early Diagnosis of Malignancy". Clin. Chemistry, 41, 1410-13.
6. Peng Liang, and Arthur B. Pardee (1992). "Differential Display of Eukaryotic Messenger RNA by Mean of the Polymerase Chain Reaction .**science, 257, 967-971.
7. L. Sompayrac, S. Jane, T.C. Burn, D.G.Tenen, and K.J. Danna(1995) .Overcoming Limitation of the mRNA Differential Display Technique. Nucleic Acid Research, 23(22) 4738-4739.
8. 許呈安 ,1998, 甲狀腺腫瘤生物分子標幟之研究.
9. Ling Chen, John P. O,Bryan, Helene S. Smith, and Edison
Liu(1990).Overexpression of Matrix Gla Protein mRNA in
Malignant Human Breast Cells: Isolation by Differential
cDNA Hybridization . Oncogene 5, 1391-1395.
10. Eleni N. Levedkou, Torsten G. Strohmeyer, Peter J. Effert, and Edison T. Liu(1992), "expression of the Matrix Gla Protein in Urogenital Malignanies." Int J. Cancer, 52, 534-537.
11. Hunter T: Oncoprotein networks. Cell 1997: 88: 333-
346.DeRisi J. Pen land L,Brown Po, et al: Use of a cDNA microarray to analyze gene .expression pattern in human cancer. Nature Genet 1996: 14: 457-460.13.
12. Sehgal A, Keener C, Boynton AL,et. : Characterization of C4-2 as a tumor- suppressor gene in human brain tumors. J. surg .on col 1997; 64: 102-108.
14. Sehgal A, Keener C, Boynton AL,et al: isolation and characterization of a novel gene from human glioblastoma multiform tumor tissue. Int .J .Cancer 1997; 71; 565-572.
15. Pend Liang, Lidia Averboukh, and Arthur B Pardee (1993) "Distribution and Cloning of eukaryotic mRNA by Means of differential Display: Refinnements and optimization.**
Nucleic Acid Research, 21(14), 3269-75.
16. 林齊宣(1995), 解剖學原理與實用, 合記., 台北.
17. 趙有誠. 1984), 胃腸疾病學-病態生理探討 ,合記, 台北.
18. Tortora GJ. Principle of human anatomy. Harper Collins College Publishers.Newyork,1992.
19. Lockhart-Mummery, J.P. 1927. Two hundred cases of cancer of the rectum treated by perinetal excision. Br. J. Surg. 14: 110-124.
20. dukes, C.E.. 1932. The classification of cancer of the rectum. J.pathol. 35: 323-332.
21. Zinkin, L.D. 1983, A critical review of the classification and staging of colorectal cancer. Disease of the colon and rectum 26: 37-43.
22. Davis, N. C., R. C. Newland. 1982. The reporting of the colorectal cancer: the Australian clinco-pathological staging system. Aust. NZ J.surg. 52: 395-397.
23. 林肇堂. 1983. 實用腸胃學. 茂昌. 台北.
24. 陳昌平. 王南琨. 張治平. 1997.血液學診段斷與實驗.合記. 台北.
25. Vogelstein, B., E. R. Fearon, S.R. Hamilton, S.E. Kern, A.C. Preisinger, M. Leppert, Y. Nakamura, R. White, A.M. M. Smits, and J. L. Bos. 1998. Genetic alternations during colorectal cancer development. New England J. Med. 319: 525-532.
26. 彭依宗, 吳建廷, 1986. X-光診斷學. 南山堂. 台北.
27. Lewi, H., L.H. Blumgard, D. C. carter, C. R. Gillis, D. Hole, J.G. ratcliffe, C. B. Wood, and C. S. McArdle. 1984. Pre-operative carcinomaa-embryonic antigen and survival in patients with colorectal cancer. Br. J. Surg. 7: 206-208.Martin, E. W., J. P. Minton, and L. C. Carey. 1985.
28. CEA-directed second-look surgery in the asympomatic patient after primary resection of colorectal carcinoma. Ann. Surg. 202(1): 310-317.
29. Moertel, C. G., T. R. Fleming, and J. S. Macdonald, et al. 1993. An evaluation of the carcinoembryotic antigen(CEA) test for monitoring patients with resected colon cancer. J. Amer. Med. Ass. 270(8): 943-947.
30. 許清曉. 1996. 常用臨床檢驗手冊. 藝軒. 台北.
31. Powell, S. M., G. M. Petersen, A.J. Krush, et al.. 1993. Molecular diagnosis of familial adenomatous polyposis. New England J. med. 329(27): 1982-1987.
32. Filella X., R. Molina, and J. J. Grau, et al. 1992. Prognostic value of CA19.9 levels in colorectal cancer. Ann. Surg. 216(1): 55-59.
33. Liu, C., A Woo, and M.-S. Tsao 1990. Expression of transforming growth factor -alpha in primary human colon and lung carcinomas. Br. J. Cancer 62: 425-429.
34. Luk, G. D., S.B. Baylin. 1984. Ornithine decarboxylase as a biologic marker in familial colonic polyposis. New England J. Med. 311(2): 80-83.
35. Hachford, A. W.. 1993. Biochemical markers for colorectal cancer-Diagnostic and Therapeutic Implication. Surg. Clinics of North America 73(1); 85-101
36. 劉幕台, 何瑞明. 1984. 放射腫瘤學. 南山堂. 台北.
37. gastrointestinal tumor study group . 1984. Adjuvant therapy of colon cancer: results of a prospectively randomized trial. New England J. Med. 310(12): 737-743.
38. Wolmark, N., H. Rockette, and B. Fisher, et al. 1993. The benefit of leucovorin-modulated fluorouracil as postoperative adjuvant therapy for primary colon cancer: results from National surgical Adjuvant Breast and Bowel project protocol C-03. J. Clin. Oncology 11(10): 1879-1887.
39. Moertel, C. G.. 1994. Chemotherapy for colorectal cancer. New England J.Med. 330916)1136-1142.
40. Conti, j. A., N. E. Kemeny, and L.B. Saltz, et al. 1996. Irrinotecan is an active agent in untreated patients with metastatic colorectal cancer. J Clin. Oncology 14(3): 709-715.
41. Rothenberg, M. L., J. R. Eckardt, and J.G. kuhn, et al. Phase II trial of Irinotecan in patients with progressive or rapidly recurrent colorectal cancer. J. Clin. Oncology 14(4); 1128-1135.
42. Fearn ER and Vogelsten B: A genetic model for colorectal tumorgenesis.Cell 1990,61:759-761.
43. Cole MD: The myc oncogene: Its role in transformation and differentiation. Annu Rev Gene 1986, 20: 361-384.
44. Erisman MD, Astrin SM: The myc oncogene, in Reddy EP, Shalka AM, Curran T: The Oncogene Handbook. New York, Elsevier, 1988, 341-379.
45. Armelin HA, Aremlin MCS, Kelly K et al: Functional role for c-myc in mitogenic response to platelet derived growth factor. Nature 1984, 310: 655-660.
46. Erisman MD, Rothberg PG, Diehl RE et al: Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene. Mol Cell Biol 1985, 5: 1969-1976.
47. Rothbrtg PG, Spandorfer JM, Erisman MD et al: Evidence that c-myc expression defines two genetically distinct from of colorectal adenocarcinoma. Br J Cancer 1985. 52: 629-632.
48. Erisman MD, Scott JK, Astrin SM: Evidence that the FAP gene is involved in a subset of colon cancers with a complementable defect in c-myc regulation. PRC Natl Acad Sci USA.
49. Touchot N, Chardin P, Tavitian A: Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci USA 1987, 84: 8210-8214.
50. Sweet RW, Yokoyama S, Kamata T et al: The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature 1984, 311: 273-275.
51. Reddy EP, Reynold RK, Santos E et al: A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene. Nature 1982, 300: 149-152.
52. Forrester K, Almoguera C, Han K et al: Detection of high incidence of K-ras oncogenesis. Nature 1987, 327:298-303.
53. Vogelstein B, Fearon ER, Hamilton SR et al: genetic alteration during colorectal tumor development. N Engl J Med 1988 319: 525-532.
54. Reichman A, Martin P, Levin B: Chromrsomal banding patterns in human large bowel cancer. Int J Cancer 1981, 28: 431-440.
55. Bodmer WF, Bailey CJ, Bodmer J et al: localization of gene for familial adenomatous polyposis on chromosome 5. Nature 1987, 328: 614-616.
56. Muto T, Kamiya J, Sawada T, KonishiF, Sugihara K etal: Small flat adenoma of the large bowel with special reference to its clinicopathologic features. Dis Col Rect 1985, 28:847-51.
57. Sassaki M, Okamoto M, Sato C, Sugio K et al: Loss of constitutional heterozygosity in colorectal tumors from patients with familial polyposis coli and those with non-polyposis colorectal carcinoma. Cancer res 1989, 49, 4402-4406.
58. Nishisho I, Nakawamura Y, Mitoshi Y et al: Mutations of chromosome 5 q 21 genes in FAP and colorectal cancer patients. Science 1991, 253 ; 665-669.
59. Kinzler KW, Nilbert MC, Vogelstein B et al: Identification of a gene located at chromosome 5 q 21 that is mutated in colorectal cancers. Science 1991, 251; 1366-1371.
60. Joslyn G, Carison M, Thliveris A et al: Identification of deletion mutations and three new genes at the familial polyposis locus. Cell 1991, 66: 601-613.
61. Bischoff JR, Friedman PN, Marshak DR, Prives C, Beach AD: Human p53 is phosphorylated by p60-cdc 2 and cyclin B-cdc 2. Proc Ntal Acad Sci USA 1990, 87:4766-4770.
62. Baker SJ, Markowitz S, Fearon ER, Willson JKV, Vogelstin B: Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 1990. 249: 912-915.
63. Nigro JM, Baker SJ, Preisinger AC et al: Mutation in the p53 gene occur in diverse human tumor cell types. Nature 1989, 342; 705-707.
64. Baker SJ, Fearon ER, Nigro JM et al: Chromosome 17 Deletion and p53 mutations in colorectal carcinoma. Science 19899, 244: 217-221.
65. KIKUCHI-Yanoshita R, Satomi MK, Ito S, Seki M et al: Genetic changes of both p53 alleles associated with the conversion from colorectal adenoma to early carcinoma in familial adenomatous polyposis and non-familial adenomatous polyposis patients. Cancer res 1992, 52: 3965-3971.
66. Kikuchi-Yanoshita R, Konishi M, Fukunari H, Tanaka K and Miyaki M: Loss of expression of the DCC gene during progression of colorectal carcinoma in familial adenomatous polyposis and non-familial adenomatous polyposis patients. Cancer Res 1992, 52: 3801-3803.
67. Fearon ER, Cho KR, Nigro JM et al: Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 1990, 247: 49-55.
68. Michael C. Kiefer, Diane M. Bauer, Daru Koung, Kathleen M. Hermsen, Frank R. Masiarz, and Philip J Barr (1988).The cDNA and Derived Amino Acid Sequences for Human and Bovine Matrix Gla Protein. Nucleic Acids Research, 16, 5213.
69.Leonor Cancela, Chih-Lin Hsieh, Uta Francke, and Paul A.
Price (1990), Molecular Structure Chromosome Assignment and Promoter Organization of the Human Matrix Gla Protein
Gene.The Journal of Biological Chemistry, 265, 25, 15040-15048.
70. James D. Fraser and Paul A.Price (1988).Lung, Heart and
Kidney Express High Levels of mRNA for the Vitamin K-
dependent Matrix Gla Protein. The Journal of Biological
Chemistry, 263, 23, 11033-11036.Micheal C Kiefer, Diane M Bauer, D
71. Anil Sehgal and Alton L.Boynton (1998).Application of the Differential Hybridization of AltasTM Human Expression Arrays Technique in the identification of Differentially Expressed Genes in Human Glioblastoma Multiforme Tumor Tissue .Journal of Surgical Oncology 1998;67:234-241.
72.Sadhu, K., Reed, S. I., Richardson, H., and Russel, P.Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2.proc. Natl.Acad. Sci.USA,87:5139-5143,1990.
73.Galaktionov,K.,and Beach, D. Specific activation of cdc25 tyrosine phosphatase by B-type cyclins : evidence for multiple roles of mitotic cyclins. Cell,67:1181-1194,1991
74.Nagata, A., Igarashi, M., Jinno, S., Suto, K., and Okaymama, H. An additional homology of the fission yeast cdc25+gene occurs in humans and is highly expressed in some cancer cells. New Biol.,3:959-968,1991.
75.Damiela G. ,Roberta M., Sara P., Tamara V., Luigi B., Sandro S. ,and Mauro B. Overexpression of CDC25A and CDC25B in Heat and Neck Cancers. Cancer reasearch 57,2366-2368,1997.
76.Weiguo W., You-Hong F., Bonnie Lkemp, Garrett W., and Li M .Cancer research 58 4082-4085,1998.
77.Chanseob Shim, Wei Z.,Chang Hun Rhee, and Je-Ho Lee. Profiling of Differentially Expressed Genes in Human Primary Cervical by Complementary DNA Expression Array