臺灣博碩士論文加值系統

烏腳病是台灣西南沿海特有的地方性疾病，患者初因末梢血管栓塞，而有手腳末梢冰冷、麻痺等症狀，最後則因末端組織缺乏養分而變黑、壞死，因而得名。除了典型烏腳、烏手以外，烏腳病病患還有許多併發症，如癌症與皮膚病，許多烏腳病病患最終死因是因為癌症，除此之外，有一個更重要的死因是心血管疾病。根據病理切片的結果，許多烏腳病的病患，不僅末梢血管有阻塞的現象，全身各主要器官的大動脈有著典型粥狀硬化的脂肪沉積現象，粥狀斑塊是動脈粥狀硬化主要的病理痕跡。粥狀動脈硬化便成為烏腳病另一項重要的病理變化。
流行病學的調查指出，烏腳病的病因與當地的居民飲用深井水有密切的關係，許多井水的成分均可能導致烏腳病產生，腐植酸是其中十分重要的有機酸，腐植酸經由注射導致烏尾鼠的動物實驗，已成功的說明其在烏腳病所扮演的角色。
本研究在說明腐植酸與烏腳病動脈粥狀硬化的關係，利用西方點墨法，證明了腐植酸可以增加血球前驅細胞 (HL-60) 脂肪過氧化體增生活化受體 (PPAR gamma) 的產生，此為一個與脂肪的代謝有關的轉錄因子，其在血球細胞的表現一般認為與血球細胞的分化有關。尤其在吞噬細胞的大量表現，可以誘導氧化型低密度脂蛋白受體(scavenger receptor CD36)的表現，使吞噬細胞成功的分化成泡沫細胞、與動脈硬化的病理機轉可以說密不可分，實驗中顯示不論是高濃度的腐植酸(0.1-0.4mg/ml)或低濃度的腐植酸(0.02-0.05mg/ml)均可單獨誘導血球前驅細胞PPARgamma的產生，且與PMA結合使用，也可加強PPARgamma的誘導作用，可見腐植酸在血球前驅細胞可作為一個PPARgamma的誘導劑。
再者，經由流式細胞儀分析的結果顯示，經腐植酸處理的血球前驅細胞，細胞表面的氧化型低密度脂蛋白受體(CD36)約增加了15倍之多，同時，與PMA結合的作用，也可以使已分化單核球上受體的數目增加約兩倍，可見腐植酸對於巨噬細胞形成泡沫細胞有明顯的影響，也可藉由此結果約略說明了腐植酸與烏腳病病患動脈粥狀硬化的相關性。

Black-foot disease is a chronic peripheral vascular disease prevalent in the western-southern Taiwan. Epidemiological studies have shown that there is a high correlation between this disease and humic acid, a fluorescent, deep brown organic acid which is easily found in various waters . Humic acid has been implicated as an etiological factor of the thromboangitic and atherothrombotic vasculopathy of the disease.
Clinical studies and microscopy examinations reveal that the signs and symptoms of Black-foot disease are similar to that of atherosclerosis , which is the progressive accumulation of smooth muscle cells and lipids with in the arterial intima. The insulation of plasma lipid into intima cells occurred by direct passage of simple lipid molecules across the endothelium and via macrophage that engulfs ox-LDL (low density lipoprotein ) in the blood or in the intima .Therefore , macrophage uptakes of ox LDL is thought to play a central role in the foam cell formation and the pathogenesis of atherosclerosis and Black-foot disease.
PPARgamma (peroxisome proliferatior-activated receptor-g ) is a nuclear receptor that forms a heterodimeric DNA binding complex with the retinoic acid receptor a (RXRa) , and serves as a transcription regulator of gene involved in adipocyte lipid metabolism. PPARgamma has been reported to be expressed in several myeloid leukemia cell lines. Recently , PPARgamma has been found expressed at high level in the foam cells of atherosclerotic lesion. Ligand activation of PPARg:RXRa heterodimer in myelocytic cell line induces change of characteristic of monocytic differentiation and promotes the uptake of ox LDL by regulating the expression of the scavenger receptor, CD36.PPARgamma and its congers may play an unexpected role in Black-foot disease.
We have found that humic acid can induce PPARgamma production in pre-adipocyte. The finding led us to search for its induction of PPARgamma in blood cells.Western Blotting result reveals the ability of humic acid induction of PPARgamma in promyelocytic leukemia cells (HL-60) .HL-60 cells were treated for 24 hours with 0.020-0.4 mg/ml humic acid , and the induced amount of PPARgamma is dependent on the concentration of humic acid .
The expression of CD36 on the surface of humic acid treated with HL-60 cells was examined by flow cytometry using a monoclonal antibody to CD36.The amount of immunoreactive CD36 on HL-60 was significantly increased when cells were treated with 20mg/ml of humic acid. A strong induction of surface CD36 expression was observed following the treatment of the combination of humic acid and phorbol ester.
The above data strongly suggest that the ability of humic acid to induce formation of foam cell, and HA plays an important role in atheroscelostic pathogenesis of Black-foot disease .

總目次
圖目次
表目次
縮寫表
中文摘要1
英文摘要3
第一章、 導論 5
第一部份、腐植酸的研究背景5
第一節、烏腳病的病理變化及病理機轉5
第二節、動脈粥狀硬化的假說與泡沫細胞的生成12
第三節、腐植酸與烏腳病的相關研究16
第二部分、PPAR轉錄因子與CD36受體22
第一節、 PPAR轉錄因子 22
第二節、 氧化型低密度脂蛋白受體CD36 25
第二章 研究目的 27
第三章 材料與方法 28
第一節、材料 28
第二節、方法 30
第三節、 數據的整理35
第四章 結果43
第一節 、藥物(PMA與腐植酸)對細胞的毒性測試43
第二節、腐植酸誘導血球前驅細胞產生PPARgamm48
第三節、經由流式細胞儀分析CD36在細胞的分佈量50
第四節、觀察細胞形態上的變化55
第五章 討論58
第一節、 腐植酸濃度的範圍使用是否具生理意義？58
第二節、腐植酸單獨處理與PMA共同處理CD36表現量的的異同？CD36基因表現
是否有其它因子參與？ 60
第三節、腐植酸在細胞級所扮演的角色為何?造使細胞週期停止？61
第四節、腐植酸對循環中的單核球的作用62
第六章、結論64
參考文獻65

1. 林克炤 (1993)[ 烏腳病防治結果彙編 ]，台灣省政府衛生處，台北市。
2. 郭宗波、陳茂雄 (1969)[ 烏腳病之外科治療長期成績 ]，中華民國醫學文
獻， 台北市。
3. Shu Yeh , and How S.W. (1963) A pathological study on the
blackfoot disease in Taiwan , Report , Insitite of Pathology
, National of Taiwan University.
4. 曾文賓、侯書文、葉曙(1965)〔395名烏腳病病人之慢性砷中毒皮膚變化〕，國
立台灣大學病理學研究所研究報告, 25-71頁。
5. Pathology , Emanuel Rubin , and Farber John L.. Third
Edition , 1999 .
6. Hideki Adachi , Masafumi Tsujimoto , Hiroyuki Arai , and
Keizo Inoue . Expression cloning of a novel scavenger
receptor from Human endothelial cells , The Journal of
Biological Chemistry , 272 , 31217-31220 , 1997.
7. Allen Sean , Khan Shabeena , Tam Shui-Pang , Marlys
Koschinsky , Patricia Taylor , and Magdi Yacoub . Expression
of adhesion molecules by Lp(a): a potential novel mechanism
for its atherogenicity , The FASEB Journal , 12 , 1765-1776
, 1998.
8. Lutgen E. , Daemen M. , Kockx M. , Doevendane P. Hofker M.
Havekee L. , Wellene h. , and De Muinck ED . Atherosclerosis
in APOE*3-Leiden transgenic mice :from proliferate to
atheromatous stage , Circulation , 99 , 276-83 , 1999
9. Yancey PG. and Jerome WG. Lysosomal sequestration of free
and esterified cholesterol from oxidized low density
lipoprotein in macrophage of different species , Journal of
lipid research , 39 , 1349-1361 , 1998
10. Tseng WP. Outcome of patients with Black-foot disease , J
Formosan Medicine Association , 74 , 37-47 , 1975 .
11. Tseng WP. Effect and dose response relationship of skin
cancer and Black-foot disease with arsenic , Environmental
Health Perspectives , 19 , 109-119 , 1977.
12. Lu FJ , Yamamura Y , and Yamauchi H. Studies on fluorescent
compounds in water of a well in black-foot disease endemic
areas in Taiwan , J Formosan Medicine Association , 87 , 66-7
75 , 1988.
13. Steelink C , Mikita MA , and Wershaw RL. Characterization
of functional groups in humic and fulvic acids , Books of
Abstracts of the 181st ACS National Meeting , Atlanta ,
Georgia.
14. Jan IR. , Chen JY. , Lu FJ. , and Ling KH. Studies on
fluorescent compounds of drinking water in Black-foot
disease endemic areas , J Formosan Medicine Association , 77
, 361-363 , 1978.
15. 呂鋒洲，蔡孟惠，林國煌 ,(1977)〔烏腳病地區地下飲用水中螢光物質之再
檢討〕 , 台灣醫誌 , 第76卷 , 58-63頁 。
16. Lu FJ. , Tsi SC. , and Chen JY. Studies on natural
toxicants occurring in artesian well water of Black-foot
disease endemic areas in Taiwan :Chromosome aberrations in
cultured lymphocytes exposed to fluorescent compounds , 9,
49-54 , 1980.
17. Chen JY. , Lu FJ. , and Ling KH. Studies on fluorescent
compounds in drinking water in Black-foot disease endemic
areas , 5 , Effects of fluorescent compounds on protein and
nucleic acid synthesis in growing Hela cells , Chinese J
Microbiology , 10 , 115-117 , (1997).
18. Lu FJ. , Shin SR , Liu TM. , and Show SH. The effect of
fluorescent humic substance existing in the well water of
Black-foot disease endemic areas in Taiwan on prothrombin
time and activated partial thromboplastin time in vitro ,
Thrombosis Research , 57 , 747. (1990).
19. 楊新玲 (1994)〔 腐植酸誘導血液凝固的機理研究〕，台大醫學院生化學所博
士論文 (呂鋒洲教授指導) 。
20. Yang Hsin-Ling ,Hset You-Cheng , Lu Fung-Jou and Der Tsai
Horng . Humic acid reduces protein -C-activating cofactor
activity of thrombomodulin of human umbilical vein
endothelial cells , British Journal of Haematology , 101 ,
16-23 , (1998).
21. Lu FJ , and Lee YS. Humic acid : Inhibition of plasmin ,
The science of total enviroment , 11 , 135 , (1992).
22. Chiu HC. , Shin SR. ,Lu FJ. , and Yang HL . Stimulation of
endothein production in culture human endothelial cells by
fluorescent compounds associated with blackfoot disease.,
Thrombosis Research , 69, 139. (1994).
23. 呂鋒洲，劉東明，(1986)，〔烏腳病患區飲用水中螢光物質動物實驗模式〕，
台灣醫誌第85卷。第352-358頁。
24. 黃蘭如 (1991) 〔烏腳病患區井水抽取物質誘導小白鼠肝中過氧化體增生之研
究〕，台大醫學院生化學所碩士論文 (呂鋒洲教授指導) 。
25. 李雅湘 (1992) 〔腐植酸研究之新進展 〕，台大醫學院生化學所碩士論文 (
呂鋒洲教授指導) , 1992
26. 李雅湘 (1999) 〔腐植酸引起脂肪細胞分化機制之研究〕，台大醫學院生化
學所博士論文 (呂鋒洲教授指導) 。
27. Gregoire Francine M. , Smas Cynthia M. , and Sul Hei Sook.
Understanding Adipocyte Differentiation , Physiological
Reviews , 78 , 3 , 783-809 , (1998).
28. Tontonoz Peter , Nagy Laszlo, Alvarez Jacqueline G. A. ,
Thomazy Vilmos A. ,and Evans Ronald M. . PPARg promote
monocyte / macrophage differentiation and uptake of oxidized
LDL , Cell , 93 , 241-252 , (1998).
29. Ricote Mercedes, Li Andrew C. , Willson Timothy M. , Kelly
Carolyn J. , and .Glass Christopher K . The peroxisome
proliferation activated receptor-g of macrophage activation
, Nature , 391 , 79-86. (1998).
30. Spiegelman Bruce M. . PPARg in monocyte: less pain , any
gain , Cell , Vol.93 , 153-155 , (1998).
31. Nagy Laszlo , Tontonoz Peter , Alvarez Jacqueline G. A. ,
Chen Hongwu , and Evans Ronald M. . Oxidized LDL regulate
macrophage gene expression through ligand activation of
PPARgamma , Cell , 93 , 229-240 , (1998).
32. Greenwalt Dale E. , Lipsky Robert H. , Ockenhouse Christian
F. , Ikeda Hisami , Tandon Narendra N. , and Jamieson G.A. .
Membrane Glycoprotein CD36 : A review of its role in
adherence , signal transduction , and transfusion medicine ,
Blood , 80 , 5 , 1105-1115 , (1992).
33. Clemetson Kenneth J. . Platelet activation :Signal
transduction via membrane receptors , Thrombosis and
Haemostasis , 4 , 111-116. (1995).
34. Silverstein Roy l., Asch Adlam S., and Nachman Ralph L..
Glycoprotein IV mediates thrombospondin-dependent platelet
monocyte and platelet -U937 cell adhesion , Journal of
clinical Investigation , 84 , 546-552 , (1989).
35. Endemann Gerda , Lawrence W. , Stanton , Kip S. , Carmen M.
Bryant , White R. Tyler , and Andrew A. Protter . CD36 is a
receptor for oxidized low density lippprotein , Vol. 268 ,
11811-11816. (1993).
36. Stem cells , edited by CS Potten.
37. Huang Tien-Shang, Lu Fung-Jou , and Tsai Chang-Wu . Tissue
distribution of absorbed humic acids , Environmental
Geochemistry and Health , 17 , 1-4 , (1995) .
38. Essential Hematology , A. V. Hoffbrand , and J. E. Pettit.
Third Edition , 1993.