染色體終端酵素( telomerase )是一種核醣核酸蛋白酵素，主要是由蛋白質及一條RNA所組成。以自己的RNA當模版複製染色體終端序列，使染色體終端延長並穩定染色體結構。而大部分肝癌細胞均有對於終端酵素功能必須的RNA分子的表現。所以，在這個研究?堙A我們將hTR、反意hTR及INFα基因置於表現質體，經過轉染入J5肝癌細胞株中，對終端酵素進行抑制作用，並且偵測hTERT、hTR、c-myc、IFNα、p16及p27等基因的mRNA表現。
結果發現，以FhTR基因處理的細胞，有染色體終端酵素被抑制及hTR表現增強的情形，而IFNα則對終端酵素活性沒有影響。而hTERT、c-myc、IFNα、p16及p27等基因的mRNA的表現不會因終端酵素被抑制與否而影響其表現。另外，也觀察到終端酵素被抑制的細胞有基因組DNA片段化的情形，顯示部分細胞有凋亡現象。

Telomerase, a ribonucleoprotein enzyme has its own internal RNA template that elongates telomeres and stabilizes chromosome structure. The mayority of hepatoma express high levels of human telomerase template RNA ( hTR ) that is essential for cellular telomerase activity. In this study, we examined whether hTR、antisence hTR and IFNα gene had a telomerase activity inhibitory effect on J5 hepatoma cell line, through transfection via an expression vector . The expression of mRNAs for hTERT、hTR、c-myc、IFNα、p16 and p27 were also detected.
The results suggested that inhibition of telomerase activity and overexpression of hTR gene were observed in FhTR-treated J5 cells. The expression of mRNAs for hTERT、c-myc、IFNα、p16 and p27 genes were not changed whether telomerase was inhibited or not. In addition, genomic DNA fragmentation was observed in telomerase inhibited J5 cells indicating that apoptosis was undergoing in these cells.

壹、 緒論
一、 染色體端節與染色體終端酵素---------1
二、 染色體終端酵素( telomerase )與癌症的關係------------------------------------------10
三、 與人類終端酵素相關的一些基因-------12
四、 染色體終端酵素的抑制---------------14
五、 干擾素α（interferon α）----------15
六、 肝癌-------------------------------16
貳、 研究目的---------------------------18
參、 材料與方法
一、 勝任細胞（competent cell）的製備---19
二、 聚合酵素連鎖反應 ( polymerase chain reaction, PCR )-----------------------------19
三、 pMSCV-neo及pMSCV-puro質體的製備----21
四、 具有IFNα、FhTR及RhTR基因重組質體的製備---------------------------------------------23
五、 細胞培養---------------------------27
六、 生長曲線及抗藥性測試---------------30
七、 轉染細胞（transfection）-----------31
八、 利用PCR方法驗證是否轉染成功--------32
九、 終端酵素活性（telomerase activity）的偵測------------------------------------------32
十、 反轉錄聚合酵素連鎖反應( reverse transcriptase-polymerase chain reaction，RT-PCR )---------------------------------------------34
十一、 Genomic DNA fragmentation 測試-----41
肆、 結果-------------------------------42
伍、 討論-------------------------------60
陸、 結論-------------------------------64
柒、 參考文獻---------------------------65

Akiyama M., Iwase S., Horiguchi-Yamada J., Saito S., Furukawa Y., Yamada O., Mizoguchi H., Ohno T., Yamada H. Interferon-alpha repressed telomerase along with G1-accumulation of Daudi cells. Cancer Lett 1999, 142: 23 – 30.

Autexier C., Pruzan R., Funk W.D., Greider C.W. Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA. EMBO J. 1996, 15: 5928 - 5935.

Bisoffi M., Chakerian A.E., Fore M.L., Bryant J.E., Hernandez J.P., Moyzis R.K., Griffith J.K. Inhibition of human telomerase by a retrovirus expressing telomeric antisense RNA. Eur. J. Cancer 1998, 34: 1242 – 1249.

Blain S.W., Montalvo E., Massague J. Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 with cyclin A-Cdk2 and cyclin D2-Cdk4. J Biol Chem 1997, 272: 25863-25872.

Brouty-Boye D., Zetter B.R. Inhibition of cell motility by interferon. Science 1980, 208 : 516 – 518.

Cerni C. Telomeres, telomerase, and myc. An update. Mut. Res. 2000, 462: 31 – 47.

Counter C.M., Hirte H.W., Bacchetti S., Harley C.B., Telomerase activity in human ovarian carcinoma. Proc. Natl. Acad. Sci. USA 1994, 91: 2900 - 2904.

Counter C.M., Hahn, W.C., Wei W., Caddle S.D., Beijersbergen R.L., Lansdorp P.M., Sedivy J.M., and Weinberg R.A. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc. Natl. Acad. Sci. U S A 1998, 95: 14723 - 14728.

Feng J., Funk W.D., Wang S.S. Weinrich S. L., Avilion A.A., Chiu C.P., Adams R.R., Chang E., Allsopp R.C., Yu J., Le S., West M.D., Harley C.B., Andrews W.H., Greider C.W., and Villeponteau B. The RNA component of human telomerase. Science 1995, 269: 1236 – 1241.

Golovleva I., Kandefer-Szerszen M., Beckman L., Lundgren E. Polymorphism in the interferon-alpha gene family. Am..J. Hum. Genet. 1996, 59: 570 – 578.

Greider C.W., Blackburn E.H. Telomeres, telomerase and cancer. Scientific American 1996, 274: 80 – 85.

Greider C.W., Blackburn E.H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985, 43: 405 –413.

Gutterman J.U., Blumenschein G.R., Alexanian R., Yap H.Y., Buzdar A.U., Cabanillas F., Hortobagyi G.N., Hersh E.M., Rasmussen S.L., Harmon M., Kramer M., Pestka S. Leukocyte interferon-induced tumor regression in human metastatic breast cancer, multiple myeloma, and malignant lymphoma. Ann Intern Med 1980,93 : 399 – 406.

Harley C.B., Futcher A.B., Greider C.W. Telomeres shorten during ageing of human fibroblast. Nature 1990, 345: 458 – 460.

Harrington L., McPhail T., Mar V., Zhou W., Oulton R., Program A. EST., Bass M.B., Arruda I., Robinson M.O. A mammalian telomerase
-associated protein. Science 1997, 275: 973 – 977.

Hastie N.D., Dempster M., Dunlop M.G., Thompson A.M., Green D.K., allshire R. C. Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990, 346: 866 – 868.

Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 1965, 37: 614 – 636.

Herbert B., Pitts A.E., Baker S.I., Hamilton S.E., Wright W.E., Shay J.W., Corey D.R. Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. U S A 1999, 96: 14276-14281.

Hiyama E., Yokoyama T., Tatsumoto N., Hiyama K., Imamura Y., Murakami Y., Koddama T., Piatyszek M.A., Shay J.K., Matsuura Y. Telomerase activity in gastric cancer. Cancer Res. 1995, 55: 3258 – 3262.

Hiyama E., Kodama T., Shinbara K., Iwao T., Itoh M., Hiyama K., Shay J.W., Matsuura Y., Yokoyama T. Telomerase activity is detected in pancreatic cancer but not in benign tumors. Cancer Res. 1997, 57: 326 – 331.

Holt S.E., Shay J.W. Role of telomerase in cellular proliferation and cancer. J. Cell Physiol. 1999, 180: 10 – 18.

Huffman K.E., Levene S.D., Tesmer V.M., Shay J.W., Wright W.E. Telomere shortening is proportional to the size of the 3’G-rich telomeric overhang. J Biol Chem. 2000 as 10.1074/jbc.M002843200.

Izbicka E., Wheelhouse R.T., Raymond E., Davidson K.K., Lawrence R.A., Sun D., Windle B.E., Hurley L.H., Von Hoff D.D. Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells. Cancer Res 1999, 59: 639-644.

Kamb A., Gruis N.A., Weaver-Feldhaus J. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264: 436 – 440.

Kim N.W., Piatyszek M.A., Prowse K.R., Harley C.B., West M.D., Ho P.L., Coviello G.M., Wright W.E., Weinrich S.L., Shay J.W. Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266: 2011 – 2015.

Kipling D., Cooke H.J. Hypervariable ultra-long telomeres in mice. Nature 1990, 347: 400 – 402.

Klobutcher L.A., Swanton M.T. ,Donini P., Prescott D.M. All gene-sized DNA molecules in fore species of hypotrichs have the same terminal sequence and an unusual 3’ terminus. Proc. Natl. Acad. Sci. USA 1981, 78: 3015 – 3019.

Kojima H., Yokosuka O., Imazeki F., Saisho H., Omata M. Telomerase activity and telomere length in hepatocellular carcinoma and chronic liver disease. Gastroenterology 1997, 112: 493 – 500.

Kondo S., Tanaka Y., Kondo Y., Hitomi M., Barnett G.H., Ishizaka Y., Liu J., Haqqi T., Nishiyama A., Villeponteau B., Cowell J.K., and Barna B.P. Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation. FASEB J. 1998, 12: 801 – 811.

Kondo S., Kondo Y., Li G., Silverman R.H., Cowell J.K. Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA. Oncogene 1998, 16: 3323 – 3330.

Kushner D.M., Paranjape J.M., Bandyopadhyay B., Cramer H., Leaman D.W., Kennedy A.W., Silverman R.H., Cowell J.K. 2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells. Gynecol. Onco.l 2000, 76: 183-192.

Kyo S., Kanaya T., Ishikawa H., Ueno H., Inoue M. Telomerae activity in gynecological tumors. Clin. Cancer Res. 1996, 2: 2023 – 2028.

Lee H.W., Blasco M.A., Gottlieb G.J., Horner J.W., Greider G.W., and DePinho R.A. Essential role of mouse telomerase in highly proliferative organs. Nature 1998, 392: 569 - 574.

Lin Y., Miyamoto H., Fujinami K., Uemura H., Hosaka M., Iwsaski Y., Kubota Y. Telomerase activity in human bladder cancer. Clin. Cancer Res. 1996, 2: 929 – 932.

Liu J.P. Studies of the molecular mechanisms in the regulation of telomerase activity. FASEB J. 1999, 13: 2091 – 2104.

Lustig A.J. Crisis intervention: The role of telomerase. Proc. Nat.l Acad. Sc.i U S A 1999, 96: 3339 – 3341.

McClintock B. The stability of broken ends of chromosome in Zea mays. Genetics 1941, 41: 234 – 282.

Morin G.B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 1989, 59: 521 – 529.

Muniyappa K., Kironmai K.M. Telomere structure, replication and length maintenance. Crit. Rev. Biochem. Mol. Biol. 1998, 33: 297-336.

Naka K., Yokozaki H., Yasui W., Tahara H., Tahara E., Tahara E. Effect of antisense human telomerase RNA transfection on the growth of human gastric cancer cell lines. Biochem. Biophys. Res. Commun. 1999, 255: 753 - 758.

Nakayama J.I., Saito M., Nakamura H., Matsuura A., Iskikawa F. TLP1: a gene encoding a protein component of mammalian telomerase is a novel member of WD repeats family. Cell 1997, 88: 875 – 884.

NaKayama J.I., Tahara H., Tahara E., Saito M., Ito K., Nakamura H., Nakanishi T., Tahara E., Ide T., Ishikawa F. Telomerase activity by hTERT in human normal fibroblasts and hepatocellular carcinomas. Nature genetics 1998, 18: 65 – 68.

Niida H., Matsumoto T., Satoh H., Shiwa M., Tokutake Y., Furuichi Y., and Shinkai Y. Severe growth defect in mouse cells lacking the telomerase RNA component. Nat. Genet. 1998, 19: 203 – 206.

Norton J.C., Piatyszek M.A., Wright W.E., Shay J.W., Corey D.R. Inhibition of human telomerase activity by peptide nucleic acids. Nat. Biotechnol 1996, 14: 615 – 619.
Ogura H., Tani K., Ozawa K., Nagata S., Asano S., Takaku F. Implantation of genetically manipulated fibroblasts into mice as antitumor alpha-interferon therapy. Cancer Res. 1990, 50: 5102 – 5106.

Oh S., Song Y.H., Yim J., Kim T.K. Identification of Mad as a repressor of the human telomerase (hTERT) gene. Oncogene 2000, 19: 1485 – 1490.

Ohmura H., Tahara H., Suzuki M., Ide T., Shimizu M., Yoshida M.A., Tahara E., Shay J.W., Barrett J.C., Oshimura M. Restoration of the cellular senescence program and repression of telomerase by human chromosome 3. Jpn J Cancer Res 1995, 86: 899 – 904.

Okamoto A., Demetrick D.J., Spillare E.A., Hagiwara K., Hussain S.P., Bennett W.P., Forrester K., Gerwin B., Serrano M., Beach D.H. Mutations and altered expression of p16INK4 in human cancer. Proc. Natl. Acad. Sci. U S A 1994, 91: 11045 –11049.

Olovnikov A.M. Principle of marginotomy in template synthesis of polynucleotides. Dokl. Akad. Nauk SSSR 1971, 201: 1496-1499.

Pitts A.E., Corey D.R. Inhibition of human telomerase by 2'-O-methyl-RNA. Proc. Natl. Acad. Sci. U S A 1998, 95: 11549-11554.

Quan S., Seiter K., Feldman E., Yang L., Argani I., Farley T.J., Abraham N.G., Ahmed T. Human CD34+ hematopoietic cells transduced by retrovirus-mediated interferon alpha gene maintains regeneration capacity and engraftment in NOD/SCID mice. Exp. Hematol. 1999, 27: 1511 – 1518.

Quesada J.R., Reuben J., Manning J.T., Hersh E.M., Gutterman J.U. Alpha interferon for induction of remission in hairy-cell leukemia N. Engl. J. Med 1984, 310 : 15-18.

Reichman T.W., Alleanell J., Wang X., Moore M.A.S., Studzinski G.P. Downregulation of telomerase activity in HL60 cells by differentiating agents is accompanied by increased expression of telomerase-associated protein. J. Cellular Bioche. 1997, 67: 13 – 23.

Roy J., Fulton T. B., Blackburn E.H. Specific telomerase RNA residues distant from the template are essential for telomerase function. Genes Dev. 1998, 12: 3286 – 3300.

Serrano M., Hannon G.J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993, 366: 704-707.

Shammas M.A., Simmons C.G., Corey D.R., Reis R.J. Telomerase inhibition by peptide nucleic acids reverses 'immortality' of transformed human cells. Oncogene 1999, 18: 6191-6200.

Sherr C.J., Cancer cell cycles. Science 1996, 274:1672-1677.

Sidky Y.A., Borden E.C., Inhibition of angiogenesis by interferons: effects on tumor- and lymphocyte-induced vascular responses. Cancer Res 1987, 47: 5155 – 5161.

Strahl C., Blackburn E.H. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol. Cell Biol. 1996, 16: 53-65.

Strander H. Interferon treatment of human neoplasia. Adv. Cancer Res. 1986, 46: 1 – 265.

Tahara H., Nakanishi T., Kitamoto M., Nakashio R., Shay J.W., Tahara E., Kajiyama G., Ide T. Telomerase activity in human liver tissues: comparison between chronic liver disease and hepatocellular carcinoma. Cancer Res. 1995, 55: 2734 – 2736.

Takakura M., Kyo S., Kanaya T., Tanaka M., Inoue M. Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer. Cancer Res. 1998, 58: 1558 – 1561.

Wang J., Xie L.Y.,Allan S., Beach D., Hannon G.J. Myc activates telomerase. Genes and Dev. 1998, 1769 – 1774.

Watson J.D. Origin on concatemeric T7 DNA. Nature New Biol. 1972, 239: 197 – 201.

Weinberg R.A. Positive and negative controls on cell growth. Biochemistry 1989, 28: 8263 – 8269.

Wu K.J., Grandori C., Amacker M., Simon-Vermot N., Polack A., Lingner J., Dalla-Favera R. Direct activation of TERT transcription by c-myc. Nature genetic 1999, 21: 220 – 223.

Yi X., Tesmer V.M., Savre-Train I., Shay J.W., Wright W.E. Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels. Mol. Cell Biol. 1999, 19: 3989-3997.

Yokoyama Y., Takahashi Y., Shinohara A., Lian Z., Wan X., Niwa K., Tamaya T. Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells. Cancer Res. 1998, 58: 5406 – 5410.

Zhu J., Wang H., Bishop J.M., Blackburn E.H. Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. Proc. Nat.l Acad. Sc.i U S A 1999, 96: 3723 – 3728.

Zijlmans J.M., Martens U.M., Poon S.S., Raap A.K., Tanke H.J., Ward R.K., Lansdorp P.M. Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci USA 1997, 94: 7423-7428.

楊東和，趙有誠，唐鴻舜。肝細胞癌綜論。國防醫學。民國八十三年九月十五日。第十九卷第三期。275-278。