造血素Erythropoietin(EPO)，可在低氧情況下(例如：CoCl2的加入)，於腎臟及胎兒肝臟中產生。並且，在人類肝癌細胞株Hep G2與Hep 3B中，其EPO蛋白質及mRNA 可被誘發。而報告指出，細胞中氧氣偵測系統會受低氧或金屬鈷及H2O2的作用，而影響EPO的表現。但有趣的是，在Hep G2與Hep 3B細胞中，可以表現EPO基因，但在J5及SK-Hep-I細胞中卻缺乏EPO基因表現的能力。所以，不同分化程度的肝癌細胞，其分化程度在調控EPO基因表現上，是否扮演一個重要的角色？另一方面，可調控內生性麩胱甘肽(glutathione, GSH)合成的酵素(g-glutamylcysteine synthetase, g-GCS)是否能調節EPO基因表現也是一個重要而有趣的問題。
在此實驗中，由有幾個方向證明了內生性GSH在控制及調控EPO基因表現方面扮演重要角色。第一、使用分化程度不同的五株肝癌細胞株當作實驗模式，其內生性GSH含量也不同，依序為Hep G2 >Hep 3B >J5 >Mahlavu >SK-Hep-I細胞，並且我們也發現g-GSH重鏈單位的活性也與此有關。而這些肝癌細胞株中，只有分化程度好的Hep G2及Hep 3B有EPO基因表現，意味著其表現可能依賴於GSH，並且其EPO基因表現可能需跨過一個最低的門檻才能達到最好的表現。第二、進一步證實GSH的角色，我們加入非致死濃度的N-acetylcysteine來增加GSH的產生。我們發現，此舉可誘發EPO基因在J5及SK-Hep-I細胞中表現。後來我們更選取GCS30細胞加入比較，GCS30細胞是由γ-GCSh cDNA 永久轉染入SK-Hep-I細胞而來的，並且也已經被證實，轉染此DNA序列的GCS30細胞株中，GSH含量高過SK-Hep-I細胞；因為NAC屬外源性增加GSH，所以我們利用GCS30細胞內源性增加GSH的特點，單獨比較其與SK-Hep-I細胞之間的EPO表現量是否不同，並且於RT-PCR及EPO蛋白質表現的實驗中證實GCS30細胞株其EPO表現量比SK-Hep-I細胞為高。
綜合上述結果，我們首先證明了，除了厭氧及氯化鈷之外，內生性GSH含量在EPO基因表現方面扮演正面調控的角色，而GSH活性如何來調控EPO基因表現的機制，則尚待進一步的實驗來加以釐清。

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does “differentiation status” of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.

中文摘要..... 2
英文摘要..... 4
緒論(背景介紹) 6
材料與方法 16
結果...... 27
討論...... 45
總結...... 49
參考資料.... 50
附錄...... 56

1.Fried W, Barone-Varelas J, Barone T. (1982) The influence of age and sex on erythropoietin titers in the plasma and tissue homogenates of hypoxic rats. Exp Hematol. 10: 472-477
2.Johnson WJ, McCarthy JT, Yanagihara T, Osmundson PJ, Ilstrup DM, Jenson BM, Bowie EJ. (1990) Effects of recombinant human erythropoietin on cerebral and cutaneous blood flow and on blood coagulability. Kidney Int . 38: 919-24
3.Frenkel EP, Suki W, Baum J. (1968) Some observations on the localization of erythropoietin. Ann N Y Acad Sci . 149: 292-293
4.Gruber DF, Zucali JR, Mirand EA. (1977) Identification of erythropoietin producing cells in fetal mouse liver cultures. Exp Hematol. 5: 392-398.
5. Suzuki T, Sasaki R. (1990) Immunocytochemical demonstration of erythropoietin immunoreactivity in peritubular endothelial cells of the anemic mouse kidney. Arch Histol Cytol. 53: 121-124
6. Flake AW, Harrison MR, Adzick NS, Zanjani ED. (1987) Erythropoietin production by the fetal liver in an adult environment. Blood. 70: 542-545.
7. Fried W, Barone-Varelas J, Morley C. (1984) Factors that regulate extrarenal erythropoietin production. Blood Cells . 10: 287-304
8. Gruber DF, Zucali JR, Wleklinski J, LaRussa V, Mirand EA. (1977) Temporal transition in the site of rat erythropoietin production. Exp Hematol. 5: 399-407
9. Law ML, Cai GY, Lin FK, Wei Q, Huang SZ, Hartz JH, Morse H, Lin CH, Jones C, Kao FT. (1986) Chromosomal assignment of the human erythropoietin gene and its DNA polymorphism. Proc Natl Acad Sci U S A . 83: 6920-4
10. Powell JS, Berkner KL, Lebo RV, Adamson JW. (1986) Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization. Proc Natl Acad Sci U S A. 83: 6465-6469
11. Watkins PC, Eddy R, Hoffman N, Stanislovitis P, Beck AK, Galli J, Vellucci V, Gusella JF, Shows TB. (1986) Regional assignment of the erythropoietin gene to human chromosome region 7pter----q22. Cytogenet Cell Genet . 42: 214-218
12. Issels RD, Nagele A, Eckert KG, Wilmanns W. (1988) Promotion of cystine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetylcysteine. Biochem Pharmacol. 37: 881-888
13. Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, Chen KK, Fox GM, Martin F, Stabinsky Z, et al. (1985) Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 82: 7580-7584
14. Lai PH, Everett R, Wang FF, Arakawa T, Goldwasser E. (1986) Structural characterization of human erythropoietin. J Biol Chem. 261: 3116-3121
15. Hiromi Muta, Akihiro Funakoshi, Takashi Baba, Naohuni Uike, Hideyuki Wakasugi, Mitsuo Kozuru, and Atsuo Jimi. (1994) Gene expression of erythropoietin in hepatocellular carcinoma. Internal Medicine. 33: 427-431
16. Recny MA, Scoble HA, Kim Y. (1987) Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin. J Biol Chem. 262: 17156-17163
17. Broudy VC, Tait JF, Powell JS. (1988) Recombinant human erythropoietin: purification and analysis of carbohydrate linkage. Arch Biochem Biophys. 265: 329-336.
18. Egrie JC, Strickland TW, Lane J, Aoki K, Cohen AM, Smalling R, Trail G, Lin FK, Browne JK, Hines DK. (1986) Characterization and biological effects of recombinant human erythropoietin. Immunobiology. 172: 213-224.
19. Tsuda E, Kawanishi G, Ueda M, Masuda S, Sasaki R. (1990) The role of carbohydrate in recombinant human erythropoietin. Eur J Biochem . 188: 405-411
20. Cotes PM. (1982) Immunoreactive erythropoietin in serum. I. Evidence for the validity of the assay method and the physiological relevance of estimates. Br J Haematol . 50: 427-438.
21. Jacobs K, Shoemaker C, Rudersdorf R, Neill SD, Kaufman RJ, Mufson A, Seehra J, Jones SS, Hewick R, Fritsch EF, et al. (1985) Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature. 313: 806-810
22. Cotes PM, Canning CE, Lind T. (1983) Changes in serum immunoreactive erythropoietin during the menstrual cycle and normal pregnancy. Br J Obstet Gynaecol. 90: 304-311
23. Beguin Y, Lipscei G, Oris R, Thoumsin H, Fillet G. (1990) Serum immunoreactive erythropoietin during pregnancy and in the early postpartum. Br J Haematol . 76: 545-549.
24. Eckardt KU, Boutellier U, Kurtz A, Schopen M, Koller EA, Bauer C. Rate of erythropoietin formation in humans in response to acute hypobaric hypoxia. J Appl Physiol. 66: 1785-1988
25. Milledge JS, Cotes PM. (1985) Serum erythropoietin in humans at high altitude and its relation to plasma renin. J Appl Physiol. 59: 360-364
26. Fukuda MN, Sasaki H, Lopez L, Fukuda M. (1989) Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. Blood . 73: 84-89
27. George WJ, Briggs DW, Rodgers GM, and Fisher JW. (1977) Metabolism of erythropoietin. In kidney hormones: Erythropoietin Edited by J.W. Fisher. London Academic. Vol. II: 73-109.
28. Anagnostou A, Schade SG, Fried W. (1978) Stimulation of erythropoietin secretion by single amino acids. Proc Soc Exp Biol Med. 159: 139-141
29. Annable L., Cotes, PM., and Mussett, MV. (1972) The second international reference preparation of erythropoietin, human, urinary, for bioassay. Bull. WHO. 47: 99-112.
30. Fukushima Y, Fukuda M, Yoshida K, Yamaguchi A, Nakamoto Y, Miura AB, Harada T, Tsuchida S. (1986) Serum Erythropoietin levels and inhibitors of erythropoiesis in patients with chronic renal failure. Tohoku J Exp Med. 150: 1-15
31. Radtke HW, Claussner A, Erbes PM, Scheuermann EH, Schoeppe W, Koch KM. (1979) Serum erythropoietin concentration in chronic renal failure: relationship to degree of anemia and excretory renal function. Blood. 54: 877-884
32. Pavlovic-Kentera V, Clemons GK, Djukanovic L, Biljanovic-Paunovic L. (1987) Erythropoietin and anemia in chronic renal failure. Exp Hematol. 15: 785-789
33. Blick M, Sherwin SA, Rosenblum M, Gutterman J. (1987) Phase I study of recombinant tumor necrosis factor in cancer patients. Cancer Res . 47: 2986-2989
34. Johnson CS, Cook CA, Furmanski P. (1990) In vivo suppression of erythropoiesis by tumor necrosis factor-alpha (TNF-alpha): reversal with exogenous erythropoietin (EPO). Exp Hematol. 18: 109-113
35. Johnson CS, Keckler DJ, Topper MI, Braunschweiger PG, Furmanski P. (1989) In vivo hematopoietic effects of recombinant interleukin-1 alpha in mice: stimulation of granulocytic, monocytic, megakaryocytic, and early erythroid progenitors, suppression of late-stage erythropoiesis, and reversal of erythroid suppression with erythropoietin. Blood . 73: 678-683
36. Faquin W, Schneider TJ, and Goldberg MA. (1990) Effects of inflammatory cytokines on erythropoietin production in Hep 3B cells(abstract). Blood. 76: 142.
37. Lenfant C, Sullivan K. (1971) Adaptation to high altitude. N Engl J Med 1971. 284: 1298-1309
38. Wang GL, Semenza GL. (1993) Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood. 82: 3610-3615
39. Ehleben W, Bolling B, Merten E, Porwol T, Strohmaier AR, Acker H. (1998) Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway. Respiration physiology. 114: 25-36
40. Neumcke I, Schneider B, Fandrey J, Pagel H. (1999) Effects of pro- and antioxidative compounds on renal production of erythropoietin. Endocrinology. 140: 641-645
41. Joachim Fandrey, Stilla Frede, and Wolfgang Jelkmann. (1994) Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem. J. 303: 507-510.
42. Ziment I.(1988) Acetylcysteine: a drug that is much more than a mucokinetic. Biomed Pharmacother. 42: 513-520.
43. De Vries N, De Flora S.(1993) N-Acetyl-l-Cysteine. J Cell Biochem. 17: S270-S277.
44. Imai N, Kawamura A, Higuchi M, Oh-eda M, Orita T, Kawaguchi T, Ochi N. (1990) Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin. J Biochem. 107: 352-359
45. De Flora S, Bennicelli C, Camoirano A, Serra D, Romano M, Rossi GA, Morelli A, De Flora A. (1985) In vivo effects of N-acetylcysteine on glutathione metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds. Carcinogenesis. 6: 1735-1745
46. Corcoran GB, Wong BK. (1986) Role of glutathione in prevention of acetaminophen-induced hepatotoxicity by N-acetyl-L-cysteine in vivo: Studies with N-acetyl-D-cysteine in mice. J Pharm Exp Ther. 238: 54-61.
47. Buhl R., Holroyd KJ, Mastrangeli A., (1989) Systemic glutathione deficiency in symptom-free HIV-seropositive individuals. Lancet. 1: 1294-1297.
48. De Flora S, Rossi GA, De Flora A. (1986)Metabolic, desmutagenic and anticarcinogenic effects of N-acetylcysteine. Respiration. 50: S43-S49.
49. Cesarone CF, Scarabelli L, Giannoni P, Orunesu M. (1990) Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei. Mutat Res. 245: 157-163
50. Reed DJ. Reed DJ. (1990) Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol. 30: 603-631
51. Jia-Shing Chen (1999), Inhibitory effects of N-acetyl-L-cysteine on cell proliferation and UV induction of PCNA in CHO-K1 cells.國立清華大學碩士論文.
52. Paul P, Rothmann SA, McMahon, and Gordon AS. (1984) Erythropoietin secretion by isolated rat Kupffer cells. Exp. Hematol. 12: 825-830
53. H. Rranklin Bunn, Jie GU, L. Eric Huang, Jong-Wan Park and Hao Zhu. (1998) Erythropoietin: a model system for studying oxygen-dependent gene regulation. The journal of Experimental Biology. 201: 1197-1201
54. Freedman MH, Grunberger T, Saunders EF. (1982) Erythropoietic inhibitors in uremic serum. Clin Invest Med . 5: 237-240
55. Liu TZ, Chen PY, Chiu DTY, Wei FS, Chang KSS, and Lin KH. (1994) Detection of a novel lactate dehydrogenase isozyme and an apparent differentiation-associated shift in isozyme profile in hepatoma cell lines. Cancer letters. 87: 193-198