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研究生:楊曉琪
研究生(外文):Hsiao-Chi Yaong
論文名稱:補充葉酸對氧化劑誘發細胞程式凋亡與啟動粒線體訊息傳遞機制的影響
論文名稱(外文):Effects of Folate Supplementation on the Reactive Oxygen Species Induced Apoptosis and Mitochondria Related Signaling
指導教授:許瑞芬許瑞芬引用關係
指導教授(外文):Rwei-Fen S. Huang
學位類別:碩士
校院名稱:輔仁大學
系所名稱:食品營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:82
中文關鍵詞:葉酸7-ketocholesterol程式凋亡粒線體膜電位超氧陰離子
外文關鍵詞:folate7-ketocholesterolapoptosismitochondrial membrane potentialsuperoxide anion
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本研究以兩個細胞株 (U937及HL-60) 為實驗模式,探討補充葉酸對氧化劑誘發細胞程式凋亡與啟動粒線體訊息傳遞機制的影響。研究中採用氧化劑7-ketocholesterol (7-KC) 經由產生超氧陰離子誘發U937細胞進行細胞程式凋亡。結果顯示,U937細胞與40μg/ml 7-KC作用24小時後,程式凋亡變形縮小的細胞比例顯著增加、含低套數DNA的細胞比例也顯著提高,出現180-200 bp 的程式凋亡DNA斷片,並且活化程式凋亡執行蛋白caspase-3的活性及cytochrome c由粒線體釋放至細胞質中。7-KC對U937細胞之程式凋亡傷害於30小時處理後達到最高點,之後細胞壞死的比例相對提高,顯示程式凋亡進行至續發性的細胞壞死。補充1000或1500 μΜ的葉酸可以顯著改善因7-KC在24小時和30小時所引發的變形細胞比例,低套數染色體細胞的比例以及減緩DNA斷片的程度。經由螢光染劑rhodamine 123與細胞流式儀發現,經40μg/ml 7-KC處理24小時所產生之細胞程式凋亡細胞中的粒腺體膜電位下降。隨著補充葉酸量的增加可減少其降低程度,而以補充1500μM 葉酸影響最為顯著。同時,也可以抑制cytochrome c的釋出,但對於pro-caspase-3活化並無減緩。當7-KC作用時間延長至30小時,1500μM 葉酸補充不但可以減少cytochrome c的釋出,而且對於pro-caspase-3的活化也有降低之影響。相對於同半胱胺酸硫化內酯 (HcyT) 誘發產生而導致細胞進行程式凋亡及膜電位相關訊息傳遞,補充葉酸卻沒有顯著之影響。因此,補充葉酸對於超氧陰離子 (以7-KC處理) 誘發的程式凋亡與粒腺體訊息傳遞機制有抑制改善之作用。但對於過氧化氫 (以HcyT處理) 誘導細胞程式凋亡模式之粒腺體膜電位變化,補充葉酸並無影響。

Human promonocytic leukemia U937 cell line and human promyeloid leukemia HL-60 cell line were used to investigate the effects of folate supplementation on reactive oxygen species (ROS) induced apoptosis and mitochondria related signaling. Previous studies have suggested that 7-ketocholesterol (7-KC) was a superoxide anion generator and apoptotic promoter. As U937 cells were treated with 40μg/ml 7-KC for 24 hours, apoptosis was evidenced by the increases of apoptotic cells with hypoploid DNA contents and internucleosomal DNA fragmentation. Mitochondrial permeability in apoptotic cells was significantly decreased by 7-KC, which was accompanied with the release of cytochrome c into the cytosol. Preincubation of 7-KC treated cells with folate (1000μM or 1500μM) for 6 hours significantly reduced the proportion of apoptotic cells with hypodiploid DNA contents, and attenuated the apoptotic DNA fragmentation, by normalizing mitochondrial permeability as well as inhibiting cytochrome c release. Effect of folate supplementation in attenuating apoptotic damage and signaling persisted 7-KC treatment lasted for 30 hours. To the systems involving homocysteine thiolactone (HcyT)-induced and H2O2-mediated apoptosis, folate supplementation failed to affect the mitochondrial membrane potential and cytochrome c release. Taken together, our findings suggest that folate supplementation was effective to reduce apoptotic damage and to suppress mitochondria related signaling, particular under the condition where as superoxide was the apoptotic mediator.

第一章 前言…………………………………………………………………………….1
第二章 文獻回顧…………………………………………………………………2
一、葉酸營養………………………………………………………….……………..2
二、葉酸缺乏導致細胞程式凋亡…………………………………………………….3
三、細胞程式凋亡與訊息傳遞機制…………………………………………………3
(一) 細胞程式凋亡………………………………………………………………...3
(二) Caspase家族………………………………………………………………..4
(三) caspase-3的受質…………………………………………………………….5
(四) 調節caspase-3之訊息傳遞路徑…………………………………………….... 5
四、 氧化壓力誘發細胞程式凋亡…………….…………………………………….…6
五、 氧化壓力與粒線體誘發細胞程式凋亡之關係………………………………… 7
六、 7-ketocholesterol與同半胱胺酸硫化內酯誘發細胞程式凋亡…………………8
(一) 7 —ketocholesterol……………………………………………………………... …….8
(二) 同半胱胺酸硫化內酯………………………………………………………...8
七、葉酸的抗氧化力………………………………………………………………..8
八、研究目的………………………………………………………………………….9
第三章 實驗材料與方法…………………………………………………………….. 14
一、 實驗材料……………………………………………………………………..14
(一). 細胞株………………………………………………………………………14
(二) 試藥與儀器設備…………………………………………………………… 14
二、實驗方法……………………………………………………………………….15
(一) 細胞培養…………………………………………………………………….15
(二) 7-KC的配製……………………………………………………………... …15
(三) 細胞生長率分析…………………………………………………………. ...15
(四) 低染色體套數(hypoploid)細胞測定………………………………………..16
(五) DNA laddering斷片測定………………………….………………………16
(六) 細胞粒線體膜電位之測定………………………………………………….17
(七) 西方墨點法………………………………………………………………….17
1. 測定細胞內pro-caspase-3蛋白……………………………………………18
2 測定細胞質內cytochrome c蛋白…………………………………………..18
3. 測定細胞內β-actin 蛋白…………………………………………………...19
(八) 細胞蛋白質濃度測定……………………………………………………….20
(九) 統計分析…………………………………………………………………….20
第四章 結 果
補充葉酸對7-KC誘發U937細胞程式凋亡與啟動粒線體訊息傳遞機制的影響
一、7-KC對U937細胞生存率之影響…….……………………………………… 21
二、補充葉酸對7-KC作用後之U937細胞生存率的影響……………………...21
三、補充葉酸對7-KC誘發U937細胞程式凋亡細胞與壞死細胞的影響……...22 (一) 補充葉酸對7-KC作用24小時引發之程式凋亡細胞與
壞死細胞的影響………………………………………………………… 22
(二) 補充葉酸對7-KC作用30小時引發之程式凋亡細胞與
壞死細胞的影響……………………………………………...………….. 22
(三) 補充葉酸對7-KC作用36小時引發之程式凋亡細胞與
壞死細胞的影響 ………………………………………………………... 23
四、補充葉酸對7-KC造成U937細胞程式凋亡相關指標之影響
(一) DNA傷害指標:低套數染色體細胞之測定……………………………….23
(二) DNA laddering斷片測定…………………………………………………….24
五、補充葉酸對7-KC造成U937細胞粒線體膜電位下降之影響……………….24
六、補充葉酸對7-KC造成U937細胞caspase-3蛋白活化之影響………………25
七、補充葉酸對7-KC造成U937細胞cytochrome c由粒線體
釋出至細胞質之影響………………………………………………………….…….26
第五章 結 果:
補充葉酸對不同氧化劑所誘發細胞粒線體膜電位下降之影響
一、補充葉酸對7-KC誘發細胞粒線體膜電位下降之影響………………………28
(一) 葉酸以不同時間預培養對7-KC誘發U937細胞
粒線體膜電位下降之影響……………………………………………… 28
(二) 補充葉酸對7-KC誘發HL-60細胞粒線體膜電位下降之影響……………28
二、補充葉酸對HcyT誘發細胞粒線體膜電位下降及相關訊息之影響…………29
三、補充葉酸對H2O2誘發U937細胞粒線體膜電位下降之影響………………..29
第六章 討論
一、7-KC誘發細胞產生細胞程式凋亡之變化..………………………………… 30
二、7-KC與誘發細胞程式凋亡之訊息傳遞分子的關係…………….……………31
三、補充葉酸對7-KC誘發細胞產生細胞程式凋亡及訊息傳遞分子
之影響…………………………………………………………………………..32
四、補充葉酸對不同氧化劑所誘發細胞粒線體膜電位下降之影響………………34
(一) 補充葉酸對不同細胞株因7-KC所誘發細胞粒線體膜電位下降之影響34
(二) 葉酸不同預培養時間對細胞受7-KC處理後之細胞粒線體
膜電位變化…………………………………………………………………34
(三) 補充葉酸對HcyT及H2O2誘發細胞粒線體膜電位下降之影響………..35
五、高劑量葉酸對細胞之影響……………………………………………………..35
第七章 結論…………………………………………………………………………… 36
參考文獻………………………………………………………………………………..71
附錄……………………………………………………………………………………..77

林秀玲 (2001) 銅離子及同半胱胺酸誘發葉酸缺乏初代肝細胞之傷害機制。輔仁大學食品營養系碩士論文。
洪千雅 (2002) 補充葉酸對7-ketocholesterol誘發HL-60細胞內活性氧物種與程式凋亡之DNA傷害的影響。輔仁大學食品營養系碩士論文。
行政院衛生署 (2001) 國人營養素攝取參考量。 Pp.1-4, 台北市
Bernardi P, Scorrano L, Colonna R, Petronilli V and Lisa FD (1999) Mitochondria and cell death. Eur J Biochem 264;687-701.
Berndt C, Mopps B, Angermuller S, Gierschik P and Krammer PH. (1998) CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4(+) T cells. Proc Natl Acad Sci USA 95: 12556-12561.
Borman LS and Branda RF (1989) Nutritional folate-deficiency in Chinese hamster ovary cells. J Cell Physiol 140;335-343.
Buemi M, Marino D, Pasquale GD, Floccari F, Ruello A, Aloisi C, Corica F, Senatore M, Romeo A and Frisina N (2001) Effects of homocysteine on proliferation, necrosis and apoptosis of vascular smooth muscle cells in culture and influence of folic acid. Thromb Res 104:207-213.
Doshi SN, McDowll IFW, Moat SJ, Lang D, Newcombe RG, Kredan MB, Lewis MJ and Goodfellow J (2001) Folate improves endothelial function in coronary artery disease. Arterioscler Thromb Vasc Biol 21:1196-1202.
Ella BW and Douglas RG (1999) Caspase induce cytochrome c release from mitochondria by activating cytosolic factors. J Biol Chem 274:17484-17490.
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A. and Nagata S. (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391:43-50.
Gorman A, McGowan A and Cotter TG (1997) Role of peroxide and superoxide anion during tumor cell apoptosis. FEBS Lett 404:27-33.
Green DR and Reed JC (1998) Mitochondria and apoptosis. Science 281: 1309-1312.
Huang RFS, Huang SM, Lin BS, Hung CY and Lu HT (2002) N-acetylcysteine, Vitamin C and Vitamin E diminish homocysteine thiolactone induced apoptosis in human promyeloid cells. J Nutr 132:2151-2156.
Huang RFS, Ho YH, Lin HL, Wei JS and Liu TZ (1999) Folate deficiency induces a cell cycle-specific apoptosis in HepG2 cells. J Nutr 129:25-31.
James SJ, Basnafian GB and Miller BJ (1994) In vivo folate deficiency induces deoxynucleotide pool imbalance, apoptosis, and mutagenesis in Chinese hamster ovary cells. Cancer Res 54:5075-5080.
James SJ, Miller BJ, Basnakian AG, Pogribny IP, Marta P and Muskhelishvili L (1997) Apoptosis and proliferation under conditions of deoxynudeotide pool imbalance in liver of folate/methyl deficient rats. Carcinogenesis 18:287-289.
John LV, Walsh ML, Chen LB (1980) Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci USA 77:990-994.
Joshi R, Adhikari S, Patro BS, Chattopadhyay S and Mukherjee T (2001) Free radical scavenging behavior of folic acid :evidence for possible antioxidant activity. Free Radic Biol Med 30:1390-1399.
Libbus BL, Borman LS, Ventrone CH and Branda RF (1990) Nutritional folate deficiency in Chinese hamster ovary cells. Chromosomal abnormalities associated with perturbations in nucleic acid precursors. Cancer Gent Cytogenet 46:231-242.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489.
Linder MC (1985) Nutrition and metabolism of vitamins.:Nutritional biochemisitry and metabolism with clinical application (Linder MC eds), pp91-94.
Lizard G, Gueldry S, Sordet O, Monier S, Athias A and Gambert P (1998) Gluthione is implied in the control of 7-ketocholesterol—induced apoptosis, which is associated with radical oxygen species production. FASEB J 12:1651-1663.
Lizard G, Miguet C, Bessede G, Monier S, Guelday S and Gambert P (2000) Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occurring during 7-ketocholesterol induced apoptosis. Free Radic Biol Med 28:743-753.
Lizard G, Moisant M, Cordeler C, Monier S, Gambert P and Lagrost L (1997) Induction of similar features of apoptosis in human and bovine vascular endothelial cells treated by 7-ketocholesterol. J Pathol 183:330-338.
Lyons MA and Brown AJ (1999) 7-Ketocholesterol Int Biochem Cell Biol 31:369-375.
Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T and Bredesen DE (1993) Bcl-2 inhibition of neural death:Decreased generation of reactive oxygen species. Science 262:1274-1277.
Kuida K, Haydar TF, Kuan C-Y, Gu Y, Taya C, Karasuyama H, Su MS-S, Rakic P and Flavell RA (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94:325-337.
Kroemer G, Dallaporta B and Resche RM. (1998) The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: 619-642.
Machlin LJ (1991) Handbook of Vitamins. 2nd ed pp.453-490.
Mark JK and Donald WH (1994) Apoptosis mediated and thymidine prevents erythroblast destruction in folate deficiency anemia. Proc Natl Sci USA 91:4067-4071.
Martin SJ, O’Brien GA, Nishioka WK, McGahon AJ, Mahboubi A, Saido TC and Green DR. (1995) J Biol Chem 270:6425-6426.
Rimpler MM, Rauen U, Schmidt T, Moroy T and Groot H (1999) Protection against hydrogen cytotoxicity in rat-1 fibroblast provided by the oncoprotein Bcl-2:Maintenance of calcium homoeostasis is secondary to the effect of Bcl-2 on cellular glutathione. Biochem J 340:291-297.
Shougang Z, John TD, Irene EK. (2000) p38 mitogen-activated protein kinase mediates Bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide. J Biol Chem 275:25939-25948.
Steegers-Theunissem RPM, Smith SC, Steegers EAP, Guilbert LJ and Baker PN (2000) Folate affects apoptosis in human trophoblastic cells. Br J Obstet Gynaecol 107:1513-1515.
Stroes ESG, van Faassen EE, YO M, Martasek P, Boer P, Govers R and Rabelink TJ (2000) Folic acid reverts dysfunction of endothelial nitric oxide synthase. Circulation 101:948-954.
Swain RA and Clair LSt (1997) The role of folic acid in deficiency status and prevention of disease. J Farm Pract 44:138-144.
Thornberry NA and Lazebnik Y (1998) Caspases: Enemies within. Science 281: 1312-1316.
Voehringer DW, McConkey DJ, McDonnell TJ, Brisbay S and Meyn RE(1998) Bcl-2expression causes redistribution of glutathione to the nucleus. Proc Natl Acad Sci USA 90:2956-2960.
Woo M, Hakem A, Elia AJ, Hahem R, Duncan GS, Patterson BJ and Mak TW (1999) In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes. J Immunol 163:4909-4916.
Yoo HS, Moon SC, Kim ND, Park BS, Jeong MH (2000) Genistine induces apoptosis of RPE-J cells by opening mitochondrial PTP. Biochem Biophys Res Commun 276:151-156.

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