雷公藤 Tripterygium wilfordii Hook.f.屬衛矛科植物的根部。最近幾年雷公藤或其所含的內容物的抗腫瘤作用頗受重視。本實驗研究發現HL60腫瘤細胞經雷公藤抽出物的處置後，細胞型態學的觀察呈現細胞凋亡的變化、細胞存活率亦隨加藥的濃度而降低，而且DNA片斷化的表現和caspase 3活性則隨加藥的濃度而增加，以上的現象證實雷公藤抽出物具有毒殺HL60血癌細胞株的作用，並造成HL60血癌細胞株細胞凋亡。其毒殺效果和加藥的劑量呈正相關。本實驗也同時證實雷公藤抽出物無害於人體周邊血液中的單核球細胞，由於這是人體的正常細胞，故雷公藤抽出物具備抗腫瘤的藥用價值。本實驗分析Bcl-2 family蛋白質的上游基因Bcl-2及Bax的基因表現作，結果證實雷公藤抽出物可抑制HL60細胞株的Bcl-2的基因表現，而促進其Bax的基因表現，Bax/Bcl-2的比率隨加藥的劑量增加而增加。以上的結果證實經由粒腺體路徑是雷公藤抽出物造成HL60細胞株凋亡的主要機制之一。本實驗亦顯示雷公藤抽出物可抑制HL60細胞的 c-Myc基因及hTERT、TP1基因的表現，證實這是誘發HL60細胞凋亡的重要因素。在HL60細胞，c-Myc 蛋白質是hTERT基因的正向調控因子。由於端粒酶的活性主要受到hTERT的調控，所以雷公藤抽出物之抑制c-Myc基因的表現從而可抑制hTERT基因，因此降低端粒酶的活性而造成腫瘤細胞的凋亡。

Tripterygium wilfordii (T. wilfordii , TW ), a wildly used herb medicine,was tested for anticancer effect on human myeloid leukemia cells, HL60 in this study. The extract powder of T. wilfordii induced the apoptosis of HL60 cells was demonstrated by morphological change, cell viability, DNA fragmentation and caspase-3 activity. However, normal human peripheral mononuclear cells remained viable under the same treatment. The T. wilfordii induced apoptosis of HL60 cells was associated with the increased Bax gene expression and decreased Bcl-2 gene expression. In addition, the gene expression of c-Myc, and hTERT, TP1, but not TR was downregulated in TW treatedHL60 cells in dose-dependent manner. Telomerase activity in HL60 cell was inhibited by the T. wilfordii . C-Myc protein is reported as a positive regulator of hTERT gene in HL60 cells. Therefore, proto-oncogene c-myc might play an essential role in the regulation of telomerase activity in HL60cells exposed to theT. wilfordii . All the treated cells showed a decrease in telomerase activity after T. wilfordii treatment. Taken togather, these results indicate that theT. Wilfordii-induced apoptosis in HL60 is mediated through mitochondrial pathway in parallel with the decrease expression of hTERT gene.

目錄
目錄 1
圖表目錄 3
論 文 正 文 5
前言 6
實驗材料與方法 15
實驗結果 19
雷公藤抽出物對於正常細胞無毒性反應： 19
雷公藤抽出物誘發人類急性髓質性白血病細胞株HL60細胞凋亡： 19
DNA片斷化及caspase 3的活化提供了雷公藤抽出物誘發HL60細胞凋亡的進一步證據： 20
雷公藤抽出物抑制HL60細胞株的Bcl-2的基因表現而促進其Bax的基因表現： 21
雷公藤抽出物抑制HL60細胞株其hTERT、TP1的基因表現；而TR的基因表現則不受其影響： 21
雷公藤抽出物抑制HL60細胞株的c-Myc的基因表現： 22
雷公藤抽出物抑制HL60細胞株端粒酶的活性： 22
討 論 23
參考文獻 26

（1）國家中醫藥管理局中華本草編委會. 中華本草 1st edn. 上海科學技術出版社. 1999：4134-4145
（2）清朝•趙學敏 本草綱目拾遺 宏業書局有限公司 1985：264-265. ﹝3﹞Chen BJ. Triptolide, a novel immunosuppressive and anti-inflammatory agent purified from a Chinese herb Tripterygium wilfordii Hook F. Leukemia & Lymphoma 2001; 42:253-265.
﹝4﹞Qiu D, Kao PN. Immunosuppressive and anti-inflammatory mechanisms of triptolide, the principal active diterpenoid from the Chinese medicinal herb Tripterygium wilfordii Hook. f. Drugs in R & D 2003; 4:1-18.
（5）Qian SZ. Tripterygium wilfordii, a Chinese herb effective in male fertility regulation. Contraception 1987; 36:335-345.
（6）Tao X, Cush JJ, Garret M, Lipsky PE. A phase I study of ethyl acetate extract of the chinese antirheumatic herb Tripterygium wilfordii hook F in rheumatoid arthritis. Journal of Rheumatology 2001; 28:2160-2167.
（7）Ren Y, Xiong L, Wu JR. Induction of mitochondrion-mediated apoptosis of CHO cells by Tripchlorolide. Cell Research 2003; 13:295-300.
（8）Jiang MR, Li YC, Yang Y, Wu JR. C-myc degradation induced by DNA damage results in apoptosis of CHO cells. Oncogene 2003; 22:3252-3259.
（9）Chang WT, et al. Triptolide and chemotherapy cooperate in tumor cell apoptosis. A role for the p53 pathway. Journal of biological chemistry 2001; 276:2221-2227.
（10）Padua RA, et al. PML-RARA-targeted DNA vaccine induced protective immunity in a mouse model of leukemia. Nature Medicine 2003; 9:1413-1417.
（11）Dennis L, et al. Harrison’s principles of internal medicine, 16th edition. McGraw-Hill, Inc. 2005; 69:453-464.
（12）Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM. Conversion of Bcl-2 to a Bax-like death effetor by caspases. Science 1997; 278:1966-1968.
（13）Green DR, ReedJC. Mitochondria and apoptosis. Science 1988; 281:1309-1312.
（14）Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria：a primary site for Bcl-2 regulation of apoptosis. Science 1997; 275:1132-1136.
（15）Blagosklonny MV, Alvarez M, Fojo A, Neckers LM. Bcl-2 protein downregulation is not required for differentiation of multidrug resistant HL60 leukemia cells. Leukemia Research 1996; 20:101-107.
（16）Eliot HE, Borner MM, Sinha BK. Differential oncogene expression and susceptibility to apoptosis in the human leukemia HL60 cell lines: implications for etoposide resistance. Anticancer Research 1995; 15:729-733.
（17）Bouchard C, Dittrich O, Kiermaier A, Dohmann K, Menkel A, Eilers EM, Luscher B. Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter. Genes & Development 2001;15:2042-2047.
（18）Fujimoto K, Takahashi M. Telomerase activity in human leukemic cell lines is inhibited by antisense pentadecadeoxynucleotides targeted against c-myc mRNA. Biochemical & Biophysical Research Communications 1997; 241:775-781.
（19）Greenberg RA, et al. Telomerse reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation. Oncogene 1999; 18:1219-1226.
（20）Holt SE, Wright WE, Shay JW. Regulation of telomerase activity in immortal cell lines. Molecular & Cellular Biology 1996;16:2932-2939.
（21）Liu L, Saldanha SN, Pate MS, Andrews LG, Tollefsbol TO. Epigenetic regulation of human telomerase reverse transcriptase promoter activity during cellular differentiation. Genes, Chromosomes & Cancer 2004; 41:26-37.
（22）Mangano R, Piddini E, Carramusa L, Duhig T, Feo S, Fried M. Chimeric amplicons containing the c-myc gene in HL60 cells. Oncogene 1998; 17:2771-2777.
（23）Vonderheide RH, Hahn WC, Schuitze JL, Nadler M. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic lymphocyte. Immunity 1999; 10:673-679.
（24）Hahn WC, Stewart SA, Brooks Mw, York SG, Eaton E, Kurachi A, Beijersbergen RL, Knoll JH, Meyerson M. Inhibition of telomerase limits the growth of human cancer cells. Nat Med. 1999; 5:1129-1130.
（25）Nilsson JA, Maclean KH, Pendeville H, Baudino TA, Cleveland JL. Mnt loss triggers Myc transcription targets, proliferation, apoptosis, and transformation. Molecular & Cellular Biology 2004; 24:1560-1569.
（26）Wu KI, Grandori C, Amacker M, Simon-Vermot N, Polack A, Lingner J, Dalla-favera R. Direct activation of TERT transcription by c-Myc. Nature.Genetic 1990; 21:220-224.
（27）Xu D, Gruber A, Peterson C, Pisa P. Supression of telomerase activity in HL60 cells after treatment with differentiating agents. Leukemia 1996; 10:1354-1357.
（28）Xu D, Popov N, Hou M, Wang Q, Bjorkholin, Gruber A, Menkel AR, Henriksson M. Switch from Myc/Mad to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells. Proceedings of National Academy of Sciences of the Uni ted States of America 2001; 98:3826-3831.
（29）Ruvolo PP, Deng X, Carr BK, May WS. A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis. Journal of Biological Chemistry 1997;273:25436-25442.
（30）Tao XL, Dong Y, Zhang NZ. A double-blind study of T2 (tablets of polyglycosides of Tripterygium wilfodii hook) in the treatment of rheumatoid arthritis]. [Chinese]Chung-Hua Nei Ko Tsa Chih Chinese Journal of Internal Medicine 1987; 26:399-402.
（31）Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady, KD, Wong WW. Substrate specificities of caspases family protease. The Journal of Biological Chemistnry 1997; 272:9677-9682.
（32）Chomczyski P, Sacchi, N. Single-step method of RNA isolated by acid guanidium thiocyanate-phenol-chloroform extraction. Analytic Biochemistry 1987; 162:156-159.
（33）N. W. Kim, F. Wu. Advances in quantification and characterization of telomerase activity by telomeric amplification protocol(TRAP). Nuc. Acids Res. 1997; 25:2595-2597.
（34）Tomomi Kuwana, et al. Bix, Bax, and Lipid cooperate to form supermolecular openings in the outer mitochondrial membrane. Cell 2002; 111:331-342.
（35）Koyanagi Y, Kobayashi D, Yajima T, Asanuma K, Kimura T, Sato T, Kida T, Yagihashi A, Kameshima H, Watanabe N. Telomerase activity is down regulated via decreases in hTERT mRNA but not TEP1 mRNA or hTERC during the differentiation of Leukemic cells. Anticancer Research 2000; 20:773-778.
（36）Akiyama M, Yamada O, Akita S, Urashima M, Horiguchi-Yamada J, Ohno T, Mizoguchi H, Eto Y, Yamada H. Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin, but hTERT expression overcomes it. Leukemia 2000: 14:1260-1265.
（37）Avi Ashkenazi, Vishva M.Dixit. Death Reveptors : Signal and Modulation. Science 1998; 281:1305-1308.
（38）Bert Vogelstein, Kenneth W Kinzler. Canncer genes and pathway they control. Nature Medicine 2004;10:789-799.
（39）Burbage C, Tagge EP, Harris B, Hall P, Fu T, Willingham MC, Frankel AE. Ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells. Leukemia Research 1997; 21:681-690.
（40）Henriksson M. Switch from Myc/Mad to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells. Proceedings of National Academy of Sciences of the United States of America 2001; 98:3826-3831.
（41）Jaffrezou JP, Maestre N, de Mas-Mansat V, Bezombes C, Levade T, Laurent G. Positive feedback control of neutral sphingomyelinase activity by ceramide. FASEB Journal 1998; 12:999-1006.
（42）Klapper W, Qian W, Schulte C, Parwaresch R. DNA damage transiently increases TRF2 mRNA expression and telomerase activity. Leukemia 2003; 17:2007-2015.
（43）Larsen CJ. The BCL2 gene,prototypr of a gene family that controlsrpogrammed cell death(apoptosis). Annales de Genetique 1994;.37:121-134.
（44）Lindstrom MS, Wiman KG. Role of genetic and epigenetic changes in Burkitt lymphoma. Seminars in Cancer Biology 2002; 12:381-387.
（45）Liu Y, Naumovski L, Hanawalt P. Nucleotide excision repair capacity is attenuated in human promyelocytic HL60 cells that overexpress BCL2. Cancer Research 1998; 57:1650-1653.
（46）Nakatake M, Sasaki N, Murakami-Murofushi K, Yamada O. Transient posttranslational up-regulation of telomerase activity during megakaryocytic differentiation of K562 cells. Biochemical & Biophysical Research Communications 2004; 314:1080-1085.
（47）Parker BW, Kaur G, Nieves-Neira W, Taimi M, Kohlhagen G, Shimizu T, Losiewicz MD, Pommier Y, Sausville EA, Senderowicz AM. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998; 91:458-465.
（48）Popov N, Hou M, Wang Q, Bjorkholin, Gruber A, Menkel AR, Henriksson M. Switch from Myc/Mad to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells. Proceedings of National Academy of Sciences of the Uni ted States of America 2001; 98:3826-3831.
（49）Reed JC. Apoptosis-based therapies. Nat. Rev Drug Discovery 2002; 1:111.
（50）Reichman TW, Albanell J, Wang X, Moore MA, Studzinski GP. Downregulation of telomerase activity in HL60 cells by differentiating agents is accompanied by increased expression of telomerase-associated protein. Journal of Cellular Biochemistry 1997; 67:13-23.
（51）Rudner J, Jendrossek V, Belka C. New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum. Apoptosis 2002; 7:441-447.
（52）Rezler EM, Bearss DJ, Hurley LH. Telomere and telomerase as drug targets. Currnt Opinion in Pharmacology 2002; 2:415-423.
（53）Savoysky E, Yoshida K, Ohtomo T, Yamaguchi Y, Akamatsu K, Yamazaki t, Yoshida S, Tsuchiya M. Down-regulation of telomerase activity is an early event in the differentiation of HL60 cells. Biochemical & Biophysical Research Communications 1996; 226:329-334.
（54）Sheng-Teng Huang , Rong-Cui Yang , Min-Yi Chen , Jong-Hwei S. Pang. Phyllanthus urinaria induces the Fas receptor/ligand expression and ceramide-mediated apoptosis in HL-60 cells. Life Sciences 2004; 75:339-351.
（55）Thatte U, Bagadey S, Dahanukar S. Modulation of programmed cell death by medicinal plants. Cellular & Molecular Biology 2000; 46:199-214.
（56）Vrana JA, Grant S, Dent P. Inhibition of the MAPK pathway abrogates BCL2-mediated survival of leukemia cells after exposure to low-dose ionizing radiation. Radiation Research 1999; 151:559-569.
（57）Watanabe N. Telomerase, cell immortality and cancer. Hokkaido Igaku Zasshi- Hokkaido Journal of Medical Science 2001; 76:127-132.