臺灣博碩士論文加值系統

細胞老化的機制一直是生物學者們廣泛研究的話題。目前對於細胞的老化主要有兩個假說機制，分別為1.端粒(telomere)的短少以及2.氧化損傷累積所造成的細胞老化，本研究乃以G6PD缺乏之細胞為模型進一步探討此兩機制之關聯性。G6PD缺乏症（Glucose-6-phosphate dehydrogenase deficiency），又叫蠶豆症，是世界上最普遍的酵素缺乏疾病（enzymopathy），全球大約有兩億人受影響。過去一般有關G6PD缺乏的研究主要集中在紅血球，而有核細胞則及少有探討之。在本實驗室先前的實驗已發現G6PD缺乏的細胞提早衰老（premature senescence）的現象。此外，正常與G6PD缺乏細胞間其telomere的長短並無太大差異。因此為了更進一步瞭解是何機制導致G6PD缺乏細胞的老化，於是利用stable transfection將telomerase gene送入正常與G6PD缺乏的細胞，了解其細胞老化的可能機制為何？經由stable transfection將端粒酶基因送入正常與G6PD缺乏細胞，結果發現細胞在經由送入完整端粒酶(hTERT)基因後，其細胞的培養代數均增加，甚至均超過150代，呈現細胞不死的現象。繼而分析細胞的特性後，細胞並無轉型的現象，而兩種細胞均大量表現端粒酶的活性，在分析正常與G6PD缺乏的細胞的端粒長短後發現兩種細胞的端粒變化相似，均有隨代數增加而減少的趨勢，但端粒短至一定程度後就不再隨代數增加而減少。另外，在培養的過程中發現G6PD缺乏細胞的生長仍有較正常細胞來的緩慢，故推測G6PD缺乏之細胞可能是受到較大的氧化損傷而導致其生長變慢。

There are two hypotheses about the cellular senescence. One is the shortening of telomere length, and the other is the accumulation of oxidative damage. The objective of this research is to use a glucose-6-phosphate dehydrogenase (G6PD) deficient human fibroblast cell as a model to further delineate cellular aging. G6PD deficiency is a common enzymopathy affecting over 200 million people worldwide. In the past, most work done on this disease have focused on RBC, and not so much on nucleated cells. Recently, our laboratory has found that G6PD-deficiency predisposes human fibroblasts to retarded growth and accelerated cellular senescence. To investigate the underlying mechanism of this accelerated aging phenomenon, we overexpressed the telomerase gene in normal and G6PD deficient fibroblasts. The cells with stable transfected telomerase gene expressed high telomerase activity. The transfected normal and G6PD deficient cells with high telomerase activity all reach immortality (beyond 150 passages). However, G6PD deficient cells still showed slower growth patent than normal counterpart. In terms of telomere length, both normal and G6PD deficient fibroblasts showed similar progressed telomere shortening with aging, but the shortening of telomere level off at a particular length. Therefore, these data suggest that the accelerated cellular senescence in G6PD deficient human fibroblast may be due to enhanced oxidative damage.

目 錄
頁次
指導教授推薦書
口試委員會審定書
授權書…………………………………………………………………...iii
簽署人須知……………………………………………………………....v
誌謝……………………………………………………………………...vi
目錄……………………………………………………………………..vii
圖表目錄………………………………………………………………...ix
縮寫表………………………………………………………………........x
中文摘要………………………………………………………………...xi
英文摘要………………………………………………………………..xii
第一章 序論……………………………………………………………1
第一節 G6PD缺乏症……………………………………………………1
第二節 細胞老化假說………………………………………………2
第三節 本實驗室的相關研究………………………………………3
第二章 材料與方法……………………………………………………..4
第一節 實驗材料……………………………………………………4
1. 細胞株………………………………………………………..4
2. 藥品，緩衝液，培養液及試劑……………………………..5
第二節 方法…………………………………………………………7
1. 細胞培養……………………………………………………..7
2. Soft agar assay……………………………………………7
3. Karyotyping…………………………………………………7
4. TRAP-ELISA assay……………………………………...7
5. DNA萃取…………………………………………………….8
6. 偵測telomere的長度……………………………………….9
第三章 結果…………………………………………………………….10
第一節 端粒酶(hTERT)基因對細胞生長的影響…………….…..10
第二節 端粒酶(hTERT)基因對細胞染色體的影響….…………..14
第三節 以Soft Agar Assay來確定細胞是否轉型………………..17
第四節 送入的基因對細胞端粒酶(telomerase)活性的影響……..19
第五節 細胞端粒酶(telomerase)的活性對細胞端粒(telomere)長短 的影響…………………………………………………….27
第四章 討論……………………………………………………………32
參考資料………………………………………………………………...35
附錄……………………………………………………………………...41

1. Au WY, Ma SK, Lie AK, Liang R, Cheng T, Kwong YL. Glucose-6-phosphate dehydrogenase deficiency and hematopoietic stem cell transplantation Bone Marrow Transplant. 29: 399-402; 2002
2. Ayene IS, Stamato TD, Mauldin SK, Biaglow JE, Tuttle SW, Jenkins SF, Koch CJ. Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress. J. Biol. Chem. 277: 9929-9935; 2002
3. Martini G, Ursini MV: A new lease of life for an old enzyme. BioEssays 18: 631-637; 1996
4. Chiu DTY, Liu TZ. Free redical and oxidative damage in human blood cells. J. Biomed. Sci. 4: 256-259; 1997
5. Fiorelli G, Martinez di Montemuros F, Cappellini MD. Chronic non-spherocytic haemolytic disorders associated with glucose-6-phosphate dehydrogenase variants. Baillieres Best Pract Res Clin Haematol 13: 39-55; 2000
6. Mehta A, Mason PJ, Vulliamy TJ. Glucose-6-phosphate dehydrogenase deficiency. Baillieres Best Pract Res Clin Haematol 13: 21-38 ; 2000
7. Hirono A, Beutler E. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-). Proc. Natl. Acad. Sci. USA 85: 3951-3954; 1988
8. Notaro R, Afolayan A, Luzzatto L. Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history. FASEB J 14: 485-494; 2000
9. Xu W, Wang J, Hua X, Du C. Detection of point mutations in exon 2 of the G6PD gene in Chinese G6PD variants. Chin Med Sci J 9: 20-3; 1994
10. Cai W, Cai L, Zhou D, Kuang Y, Zhou Y, Stefania F, Giuseppe M. [1376G-->T mutation of G6PD gene in Han and Li nationalities in Hainan, China.] Zhonghua Yi Xue Yi Chuan Xue Za Zhi 17: 326-328; 2000
11. Iwai K, Hirono A, Matsuoka H, Kawamoto F, Horie T, Lin K, Tantular IS, Dachlan YP, Notopuro H, Hidayah NI, Salim AM, Fujii H, Miwa S, Ishii A. Distribution of glucose-6-phosphate dehydrogenase mutations in Southeast Asia. Hum Genet 108: 445-449; 2001
12. Virginia U, David T, Steve G. Role of telomerase in cell senescence and oncogenesis. Annu. Rev. Med. 51: 65-79; 2000
13. Harley CB. Telomerase is not an oncogene. Oncogene 21: 494-502; 2002
14. Johnson FB, Sinclair DA, Guarente L. Molecular biology of aging. Cell 96: 291-302; 1999
15. Jennings BJ, Ozanne SE, Hales CN. Nutrition, oxidative damage, telomere shortening, and cellular senescence: individual or connected agents of aging? Mol. Genet. Metab. 71: 32-42; 2000
16. Carol W. Greider. Telomere length regulation. Annu. Rev. Med. 65: 337-365; 1996
17. Blackburn EH. Telomerases. Annu. Rev. Biochem. 61: 113-129; 1992
18. Shay JW, Wright WE. Telomeres and telomerase: implications for cancer and aging. Radiat Res 155: 188-193; 2001
19. Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE, Shay JW. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat Genet 21: 115-118; 1999
20. Burger AM, Fiebig HH, Kuettel MR, Lautenberger JA, Kung HF, Rhim JS. Effect of oncogene expression on telomerase activation and telomere length in human endothelial, fibroblast and prostate epithelial cells. Int J Oncol 13: 1043-1048; 1998
21. Reddel RR. Genes involved in the control of cellular proliferative potential. Ann. N. Y. Acad. Sci. 20: 8-19; 1998
22. Munro J, Steeghs K, Morrison V, Ireland H, Parkinson EK. Human fibroblast replicative senescence can occur in the absence of extensive cell division and short telomeres. Oncogene 20: 3541-3552; 2001
23. Duncan EL, Wadhwa R, Kaul SC. Senescence and immortalization of human cells. Biogerontology 1: 103-21; 2000
24. Shawn E. Holt , Jerry W. Shay. Role of telomerase in cellular proliferation and cancer. J. Cell Physiol. 180: 10-18; 1999
25. Collins K, Mitchell JR. Telomerase in the human organism. Oncogene 21: 564-79; 2002
26. Levine RL, Stadtman ER. Protein modification with aging. In: Schneider, E. L., Rowe, J. W., eds. Handbook of the biology of aging. New York: Academic Press: pp.184-197; 1996
27. Ho HY, Cheng ML, Lu FJ, Chou YH, Stern A, Liang C, DTY Chiu. Enhanced oxidative stress and accelerated cellular senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts. Free Radic. Biol. Med 29: 156-169; 2000
28. Uddhav P, Jennifer B, Cynthia Cohen, Dirk D, Thomas E, and Kamal F. Overexpression of 15-lipoxygnease-1 in PC-3 human prostate cancer cells increases tumorigenesis. Carcinogenesis 22: 1765-1773; 2001
29. Carney SA, Tahara H, Swartz CD, Risinger JI, He H, Moore AB, Haseman JK, Barrett JC, Dixon D. Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics. Lab Invest 82: 719-28; 2002
30. Nam W. Kim, Fred Wu. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res. 25: 2595-2597; 1996
31. Wei L, Guo Y, Yan Z. [Detection of human telomerase activity by telomerase TRAP-ELISA assay] Zhonghua Zhong Liu Za Zhi 20: 264-6; 1998
32. Lorenz M, Saretzki G, Sitte N, Metzkow S, von Zglinicki T. BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection. Free Radic. Biol. Med 31: 824-831; 2001
33. Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11: 1921-1929; 1992
34. Wang GL, Lo KW, Tsang KS, Chung NY, Tsang YS, Cheung ST, Lee JC, Huang DP. Inhibiting tumorigenic potential by restoration of p16 in nasopharyngeal carcinoma. Br. J. Cancer 81: 1122-1126; 1999
35. Kondoh N, Yamada T, Kihara-Negishi F, Yamamoto M, Oikawa T. Enhance expression of the urokinase-type plasminogen activator gene and reduced colony formation on soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells. Br. J. Cancer 78: 718-723; 1998
36. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352; 1998
37. Mathon NF, Lloyd AC. Cell senescence and cancer. Nature Rev. Cancer 1: 203-213; 2001
38. Sozou PD, Kirkwood TB. A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA. J. Theor. Biol. 213: 573-586; 2001
39. Macera-Bloch L, Houghton J, Lenahan M, Jha KK, Ozer HL. Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis. J. Cell. Physiol. 190: 332-344; 2002
40. Helder MN, Wisman GB, van der Zee GJ. Telomerase and telomere: from basic biology to cancer treatment. Cancer Invest. 20: 82-101; 2002
41. Stewart SA, Weinberg RA. Senescence: does it all happen at the ends? Oncogene 21: 627-630; 2002
42. Ducray C, Pommier JP, Martins L, Boussin FD, Sabatier L. Telomere dynamic, end-to-end fusions and telomerase activation during the human fibroblast immortalization process. Oncogene 18: 4211-4223; 1999
43. Yan P, Benhattar J, Coindre JM, Guillou L. Telomerase activity and hTERT mRNA expression can be heterogeneous and does not correlate with telomere length in soft tissue sacormas. Int. J. Cancer 98: 851-6; 2002
44. Weng NP, Hodes RJ. The role of telomerase expression and telomerase length maintenance in human and mouse. J. Clin. Immunol. 20: 257-267; 2000
45. Grune T, Shringarpure R, Sitte N, Davies KJ. Age-related changes in protein oxidation and proteolysis in mammalian cells. J. Gerontol. A Biol. Sci. Med. Sci. 56: B459-467; 2001
46. Stolzing A, Grune T. The proteasome and its function in the ageing process. Clin. Exp. Dermatol. 26: 566-572; 2001
47. te Poele RH, Okorokov AL, Jardine L, Cummings J, Joel SP. DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 62: 1876-1883; 2002
48. Wolf FI, Torsello A, Covacci V, Fasanella S, Montanari M, Boninsegna A, Cittadini A. Oxidative DNA damage as a marker of aging in WI-38 human fibroblasts. Exp Gerontol 37: 647-656; 2002
49. Ingrosso D, Cimmino A, D'Angelo S, Alfinito F, Zappia V, Galletti P. Protein methylation as a marker of aspartate damage in glucose-6-phosphate dehydrogenase-deficient erythrocytes: role of oxidative stress. Eur J Biochem 269: 2032-2039; 2002
50. Soti C, Csermely P. Molecular chaperones and the aging process. Biogerontology 1: 225-233; 2000
51. Liu J, Atamna H, Kuratsune H, Ames BN. Delaying brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites. Ann N Y Acad Sci 959: 133-166; 2002
52. Terman A. Garbage catastrophe theory of aging: imperfect removal of oxidative damage? Redox Rep 6: 15-26; 2001