臺灣博碩士論文加值系統

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

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