臺灣博碩士論文加值系統

研究目的：本實驗之研究目的，在探討降血脂藥物HMG CoA reductase inhibitor對人類視網膜色素上皮細胞分泌血管內皮生長因子之影響。

研究材料及方法：本實驗以培養之人類視網膜色素上細胞為對象，加入HMG CoA reductase inhibitor後，先以MTT ASSAY測試是否產生細胞毒性並觀察細胞型態上的變化，再以固定濃度之HMG CoA reductase inhibitor，Atorvastatin 1.0 μM與視網膜色素上細胞作用。以RT-PCR及electrophoresis偵測VEGF mRNA的表現，並以ELISA的方法偵測VEGF proteins濃度的變化，觀察VEGF的濃度是否會受到HMG CoA reductase inhibitor的影響。

研究結果：HMG CoA reductase inhibitor在較低濃度0.1μM和1.0μM時，24小時和48小時之後並未對視網膜色素上皮細胞產生毒性反應。10 μM濃度在24小時的時間，MTT assay的數值顯示，並沒有產生明顯細胞死亡的情形。在48小時細胞存活度明顯下降(P < 0.01)，表示10 μM濃度的HMG CoA reductase inhibitor對視網膜色素細胞，隨時間增加而產生毒性作用。加入1.0 μM濃度的HMG CoA reductase inhibitor之後，視網膜色素細胞合成VEGF mRNA 的表現有顯著下降的情形(P < 0.05)。此外，加入1.0 μM濃度的HMG CoA reductase inhibitor也會讓視網膜色素細胞分泌VEGF proteins出現顯著下降的情形(P < 0.05)。

結論：HMG CoA reductase inhibitor在體外的實驗顯示10 μM濃度時，對視網膜色素上皮細胞會產生毒性反應。在1.0 μM濃度時，會對視網膜色素上皮細胞VEGF轉錄及轉譯作用產生抑制現象，而使VEGF蛋白質合成的濃度減少。

Purpose: To determinate whether HMG CoA reductase inhibitor affects vascular endothelial growth factor secretion of human retinal pigment epithelium cells.

Material and method: Cultured human pigment epithelium cell line (R50) was applied in our study. To determine cytotoxicity of HMG CoA reductase inhibitor, MTT assay and morphological observation under microscope were undergone. We added the HMG CoA reductase inhibitor to retinal pigment epithelium cells in safe concentration of 1.0 μM to evaluate VEGF mRNA expression at certain time point by RT-PCR and electrophoresis. VEGF proteins were also detected by ELISA method after retinal pigment epithelium cells incubated with HMG CoA reductase inhibitor.

Result: At lower concentration of 0.1 and 1.0 μM HMG CoA reductase inhibitor, MTT assay showed no cytotoxicity to retinal pigment epithelium cells at time of 24 and 48 hours. MTT assay showed no cytotoxicity for 10 μM HMG CoA reductase inhibitor to RPE cells at time of 24 hours. However, markedly decreased of RPE cells survival after 48 hours of incubation (P < 0.05).
After 1.0 μM HMG CoA reductase inhibitor was added to RPE cells, VEGF mRNA expression was suppressed ( P < 0.05 ) at 30, 60 and 360 minutes compared with control. 1.0μM HMG CoA reductase inhibitor also demonstrate reduction of VEGF protein after incubated with RPE cells.

Conclusion: In vitro, HMG CoA reductase inhibitor showed marledly cytotoxicity to RPE cells at concentration of 10 μM after 48 hours. Under concentration without cytotoxicity, HMG CoA reductase inhibitor suppressed the transcription of VEGF mRNA and translation of VEGF protein from RPE cells.

目 次
中文摘要 -------------------------------------- 1
英文摘要 -------------------------------------- 3
致謝辭 ---------------------------------------- 5
I.前言 ---------------------------------------- 7
II.前人研究 ----------------------------------- 12
III.研究材料與方法 ---------------------------- 26
IV.研究結果 ----------------------------------- 37
V.討論 ---------------------------------------- 42
VI.結論 --------------------------------------- 51
VII.參考文獻 ---------------------------------- 52
VIII.附錄 ------------------------------------- 61

1. Russell WR, Lawrence PC, Narsing AR, et at al. Occlusive retinal vasculitis associated with systemic lupus erythematous. Arch Ophthalmol 2000; 118(4): 588-9.
2. Unilateral retinal vasculitis, branch retinal artery occlusion and subsequent retinal neovascularization in Crohn’s disease. International Ophthalmol 2001; 24(2): 89-92.
3. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD. VEGF in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994; 331:1480-1487.
4. Peter Carmeliet, Rakesh KJ. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249-257.
5. Peter Carmeliet. Mechanism of angiogenesis and arterogenesis. Nature Med 2000; 6: 389-395.
6. William L.Lowe Jr. The role of growth factors in the pathogenesis of diabetic microvascular complications. Diabetes mellitus 3rd edition 2004; 3: 1469-1484.
7. Napoleone F, Terri DS. The Biology of Vascular Endothelial Growth Factor. Endocrine review 18(1): 4-25, 1997.
8. Michael K, Patricia AD. Vascular endothelial growth factor and its receptors . Cytokine and growth factor review. 1996; 7(3): 257-270.
9. Tischer E, Mitchell R, Hartman T, et al. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Bio Chem 1991; 266: 11947-11954.
10. Adams AP, Miller JW, Bermal MT, et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994; 118: 445-450.
11. Pepper MS, Mandriota SJ, Vassalli JD, Orci L, Montesano R. Angiogenesis- regulating cytokines: activities and interactions. Current Top Microbiol Immunol. 1996; 213: 31-67.
12. Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359: 843-845.
13. Namiki A, Brogi E, Kearney M, et al. Hypoxia Induces Vascular Endothelial Growth Factor in Cultured Human Endothelial Cells. J boil Chem 1995; 272(52): 31189-95.
14. Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor . Invest Ophthal Vis Sci 37(9); 1929-1934, 1996.
15. Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age- related macular degeneration-related choroidal neovascular membranes. Invest Ophthal Vis Sci 37(5); 855-868, 1996.
16. Robert NF, Rajesh HA, Dean E, James EP, et al. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthal 122; 393-403, 1996.
17. Pinatel VS, Guerrin M, Chollet P, et al. Vasculotropin-VEGF Stimulates Retinal Capillary Endothelial Cells Through an Autocrine Pathway. Invest Ophthalmol Vis Sci 1994; 35:3393-3400.
18. Adamis AP, Shima DT, Yeo KT, and et al. Synthesis and Secretion of Vascular Permeability Factor/Vascular Endothelial Growth Factor by Human Retinal Pigment Epithelial Cells . Biochemical & Biophysical Research Communications. 1993; 193(2): 631-8.
19. Ishibashi T, Hata Y, Yoshikawa H, and et al. Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Grafes Arch Clin Exp Ophthalmol 1997; 235: 159-167.
20. Hunninghake DB, Knopp RH, Schonfeld G, et al: Efficacy and safety of pravastatin in patients with primary hypercholesterolemia. A dose-response study. Atherosclerosis 1990; 85: 81–9.
21. Guymer RH, Chiu AW, Lim L, Baird PN. HMG CoA Reductase Inhibitors (Statins): Do They Have a Role in Age-related Macular Degeneration? Surv Ophthalmol 2005; 50: 194–206.
22. Wolfgang Dichtl, Jozef Dulak, Matthias Frick, Hannes F. Alber, Severin P. Schwarzacher, et al. HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2003; 23: 58-63.
23. Takemoto M, Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol 2001; 21: 1712–9.
24. Bellosta S, Ferri N, Bernini F, et al. Non-lipid-related effects of statins. Ann Med 2000; 32: 164-76.
25. Plenge JK, Hernandez TL, Weil KM, et al: Simvastatin lowers C-reactive protein within 14 days: an effect independent of low-density lipoprotein cholesterol reduction. Circulation 2002; 106: 1447–52.
27. McGwin G, Owsley C, Curcio CA, Crain JC. The association between statin use and age related maculopathy. Br J Ophthal 2003; 87: 1121-1125.
28. Endo A, Kuroda M, Tsujita Y. ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinium. J Antibiot 1976; 29(12): 1346-8.
29. Frank MS, Marc AP, Lemuel AM, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Eng J Med 1996; 335(14): 1001-1009.
30. Pekkanen J, Linn S, Heiss G, et al. Ten-year mortality from cardiovascular disease in relation to cholesterol level among men with and without preexisting cardiovascular disease. Eng J Med 1990; 322:1700-1707.
31. Satoru Niwa, Tetsuya Totsuka, Shigehiro Hayashi. Inhibitory effect of fluvastatin, an HMG-CoA reductase inhibitor, on the expression of adhesion molecules on human monocyte cell line. Int J Immunopharmacol 1996; 18: 669-675.
32. Wassman S, Faul A, Benno H, et al: Rapid effect of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase inhibition on coronary endothelial function. Circ Res 2003; 93: 98–103.
33. Ulrich Laufs, Fata VL, Liao JK. Inhibition of 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Blocks Hypoxia-mediated Down-regulation of Endothelial Nitric Oxide Synthase. J Biol Chem 1997; 272: 31725-9.
34. Mendall MA, Patel P, Ballam L, et al. C Reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study. BMJ 1996; 312: 1061-1065.
35. Crouse JR, Byington RP, Furberg CD. HMG-CoA reductase inhibitor therapy and stroke risk reduction: an analysis of clinical trials data. Atherosclerosis 1998; 138: 11-24.
36. Aiello LP. Vascular endothelial growth factor: 20th-century mechanisms, 21st-century therapies. Invest Ophthalmol Vis Sci 1997; 38: 1647-52.
37. Senger DR, Connolly DT, Van de Water L, Feder J, Dvorak HF. Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. Cancer Res 1990; 50: 1774-8.
38. Keck PJ, Hauser SD, Krivi G, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989; 246: 1309-12.
39. Ferrara N, Houck KA, Jakeman LB, Winer J, Leung DW. The vascular endothelial growth factor family of polypeptides. J Cell Biochem 1991; 47: 211-8.
40. Plate KH, Breier G, Welch HA, Risau W. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature 1992; 359: 845-8.
41. Witmer AN, Vrensen GF, Van Noorden CJ, Schlingemann RO. Vascular endothelial growth factors and angiogenesis in eye disease. Prog Retin Eye Res 2003; 22: 1-29.
42. Neufeld G, Cohen T, Gitay-Goren H, et al. Similarities and differences between the vascular endothelial growth factor (VEGF) splice variants. Cancer Metastasis Rev 1996; 15: 153-158.
43. Thieme H, Aiello LP, Takagi H, Ferrara N, King GL. Comparative analysis of vascular endothelial growth factor receptors on retinal and aortic vascular endothelial cells. Diabetes 1995; 44: 98-103.
44. McLeod DS, Taomoto M, Cao J, Zhu Z, Witte L, Lutty GA. Localization of VEGF receptor-2 (KDR/Flk-1) and effects of blocking it in oxygen-induced retinopathy. Invest Ophthalmol Vis Sci 2002; 43: 474-82.
45. Khawly JA, Saloupis P, Hatchell DL, Machemer R. Daunorubicin treatment in a refined experimental model of proliferative vitreoretinopathy. Grafe’s Arch Exp Oph 1991; 229:464-467.
46. Miller JW, Adamis AP, Shima DT, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol 1994;145:574-84.
47. Donahue ML, Phelps DL, Watkins RH, LoMonaco MB, Horowitz S. Retinal vascular endothelial growth factor (VEGF) mRNA expression is altered in relation to neovascularization in oxygen induced retinopathy. Curr Eye Res 1996; 15: 175-84.
48. Araiz JJ, Refojo MF, Arroyo MH, et al. Antiproliferative effect of retinoic acid in intravitreous silicone oil in an animal model of proliferative vitreoretinopathy. Invest Oph Vis Sci 1993; 34: 522-530.
49. Blumenkranz MS, Ophir A, Claflin AJ, Hajek A. Fluorouracil for the treatment of massive periretinal proliferation. Am J Ophthalmol 1982; 94: 458-467.
50. Frank RN, Amin RH, Eliott D, Puklin JE, Abrams GW. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol 1996; 122: 393-403.
51. Adamis AP, Shima DT, Tolentino MJ, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol 1996; 114: 66-71.
52. Robinson GS, Pierce EA, Rook SL, Foley E, Webb L, Smith LE. Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc Natl Acad Sci USA 1996; 93: 4851-6.
53. Amin R, Puklin JE, Frank RN. Growth factor localization in choroidal neovascular membranes of age-related macular degeneration. Invest Ophthalmol Vis Sci 1994; 35: 3178-88.
54. Yi X, Ogata N, Komada M, et al. Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol 1997;235: 313-9.
55. Campochiaro PA. Retinal and choroidal neovascularization. J Cell Physiol 2000; 184: 301-10.
56. Kwak N, Okamoto N, Wood JM, Campochiaro PA. VEGF is major stimulator in model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2000; 41: 3158-64.
57. Ronnie TP, Sheung TF, John Wong. Clinical Implications of Circulating Angiogenic Factors in Cancer Patients. J Clin Onco 2001; 19: 1207-25.
58. Kaushik S, Anoop M, Atul K, et al. Simvastatin retards progression of retinopathy in diabetic patients with hypercholestrolemia. Diabetes Research and Clincal Practice 2002; 56: 1-11.
59. Delcourt C, Michel F, Colvez A, et al. Associations of cardiovascular disease and its risk factors with age-related macular degeneration: the POLA study. Ophthalmic Epidemiol 2001; 8: 237-249.
60. Klein R, Klein BE, Jensen SC, et al. Medication use and the 5-year incidence of early age-related maculopathy. Arch Ophthalmol 2001; 119: 1354-9.
61. Napoleone Ferrara. Vascular Endothelial Growth Factor: Basic Science and Clinical Progress. Endocrine Rev 2004; 25: 581-611.
62. Adams AP, Shima DT. The role of vascular endothelial growth factor in ocular health and disease. Retina 2005; 25(2): 111-8.
64. Wijngaarden PV, Coster DJ, Williams KA. Inhibitors of Ocular Neovascularization: Promises and Potential Problems. JAMA 2005; 293: 1509-13.
68. TAP and VIP Report No. 2. Photodynamic Therapy of Subfoveal Choroidal Neovascularization With Verteporfin: Fluorescein Angiographic Guidelines for Evaluation and Treatment. Arch Ophthalmol 2003; 121: 1253-68.
69. Michels S, Rosenfeld PJ, Puliafito CA, et al. Systemic Bevacizumab (Avastin) Therapy for Neovascular Age-Related Macular Degeneration: Twelve-Week Results of an Uncontrolled Open-Label Clinical Study. Ophthalmology 2005; 112: 1035-47.
70. Nguyen QD,Shah S, Tatlipinar S,et al. Bevacizumab suppresses choroidal neovascularisation caused by pathological myopia. Br J Ophthalmol 2005; 89: 1368-70.
71. Avery RL, Pieramici DJ, Rabena MD, et al. Intravitreal Bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmology 2006; 113: 363-372.
72. Marcia Barinaga. Cancer research: From Bench Top to Bedside. Science, 278: 1036-1039, 1997.
73. Fenton RG, Kun H, Longo DL, Smith MR. Regulation of intracellular actin ploymerization by prenylated cellular proteins. J Cell Biol 1992; 117: 347-356.
74. Pérez-Sala D, Mollinedo F. Inhibition of isoprenoid biosynthesis induces apoptosis in human promyelocitic HL-60 cells. Biochem Biophys Res Commun 1994; 199: 1209-1215.
75. Carmen C, Antonio P, Maria P and et al. Role of inhibitors of isoprenylation in proliferation, phenotype and apoptosis of human retinal pigment epithelium. Graefe’s Arch Clin Exp Ophthalmol 2001; 239: 188-198.
76. Blanco-Colio LM, Villa A, Ortego M, et al. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation. Atherosclerosis 2002; 161: 17-26.
77. 陳昶齡, 高英賢, 吳文權. Hypoxia Regulation of Vascular Endothelial Growth Factor in Retinal Pigment Epithelial Cell. 2002中華民國眼科醫學會年會。
78. Shima D, Adamis AP, Yeo KT, et al. Hypoxic regulation of vascular permeability factor (vascular endothelial growth factor) mRNA and protein secretion by human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1993; 34: 900. abstract.
79. Adamis AP, Miller JW, O''Reilly M, et al. Vascular permeability factor (vascular endothelial growth factor) is produced in the retina and elevated levels are present in the aqueous humor of eyes with iris neovascularization. Invest Ophthalmol Vis Sci 1993; 34: 1440-1440. abstract.
80. Hall NF, Gale CR, Syddall H, et al. Risk of macular degeneration in users of statins: cross sectional study. BMJ 2001; 323: 375–6.
81. McCarty CA, Mukesh BN, Guymer RH, et al. Cholesterol-lowering medications reduce the risk of age-related maculopathy progression. MJA 2001; 175: 340.