臺灣博碩士論文加值系統

真核細胞中約有80 %的蛋白質分解都是透過ubiquitin-proteasome路徑，所以蛋白酶體 (proteasome) 的活性對於維持細胞代謝平衡是很重要的。蛋白酶體主要的功能為分解細胞中摺疊錯誤、半衰期較短以及表現量過多的蛋白質。現今的研究顯示在癌細胞中抑制蛋白酶體的活性，可造成細胞週期停滯、抑制細胞增生、誘導細胞內的ROS (Reactive oxygen species) 的增加以及細胞凋亡，但是目前仍有許多機制是不清楚的，所以我們想藉由探討蛋白酶體抑制劑MG132誘導白血病細胞K562凋亡之機制，進而了解蛋白酶體的作用機制。
在我們的研究顯示，K562細胞在1.4 μM MG132長時間 (48小時) 作用下，會促使細胞停留在G2/M phase、抑制細胞增生、誘導RONS (Reactive oxygen nitrogen species) 產生、造成粒線體膜電位 (ΔΨm) 受損以及導致caspase-dependent細胞凋亡；另外在MG132的作用下，可誘導細胞內iNOS (inducible Nitric oxide synthases) 增加以及些微降低抗氧化酵素MnSOD ( Manganese superoxide dismutase) 的含量。利用3 mM AG (Aminoguanidine, iNOS抑制劑) 以及3 mM NAC (N-acetyl cysteine, ROS抑制劑) 分別與1.4 μM MG132一起作用24以及48小時，確認RONS對於K562細胞的影響；由結果可知，在AG作用下藉由減少iNOS以及增加MnSOD的表現量，可抑制RONS的產生，並降低ΔΨm受損以及細胞凋亡的比率；由此可知MG132可以透過增加iNOS的表現量導致細胞內RONS的增加，進而促使細胞凋亡。

In the eukaryotic cell, about 80% of protein degradation is through ubiquitin-proteasome pathway. Proteasome activity is very important on the maintenance of cell homeostasis. The function of proteasome is to hydrolyze and degrade unfolded, short half-life and excessive proteins. Inhibitors of proteasome significantly display the therapeutic effect on cancers. Previous studies showed that inhibition of proteasome activity can cause cell cycle arrest, inhibition of cell proliferation, induction of intracellular ROS (Reactive oxygen species) and apoptosis. However, the mechanisms are largely unknown. Therefore, we used proteasome inhibitor, MG132, to investigate the mechanisms in leukemic K562 cells. The results showed that MG132 induced the G2/M arrest and inhibited cell proliferation when incubated for 48 hours. Besides, MG132 increased RONS (Reactive oxygen nitrogen species) generation (including superoxide and nitric oxide), mitochondrial membrane potential (ΔΨm) damage, and caspase-dependent apoptosis. Furthermore, MG132 induced iNOS (inducible Nitric oxide synthases) and reduced Mn-SOD (Manganese superoxide dismutase) expression. To confirm the effects of RONS, we treated K562 cells with 3mM AG (Aminoguanidine is an inhibitor of iNOS) or 3mM NAC (N-acetyl-L-cysteine is an inhibitor of ROS) together with MG132 for 24 and 48 hours. The results indicated that AG or NAC inhibited the effects of MG132 and reduced the levels of RONS, iNOS, ΔΨm damage and apoptosis. In summary, these results suggested that MG132 may have the therapeutic effect through increased iNOS and decreased Mn-SOD expression level, causing the intracellular redox imbalance, and thereby promoting apoptosis of K562 cells.

中文摘要……………………………………………………………………..………Ⅰ
Abstract………………………………………………………………………………Ⅱ
目錄…………………………..……………………………………………………....III
縮寫檢索表……………………..………………..…………………………………VII
緒論…………………………..………………………………………………………..1
蛋白酶體……………………………………………………………………………1
蛋白酶體抑制劑……………………………………………………………………2
蛋白酶體抑制劑作用機制…………………………………………………………3
A. 誘導ER stress………………………………………………………………..…3
B. 抑制NF-κB活性…………………………………………………………..……3
C. 誘導細胞週期停…………………………………………………..……..……..4
D. 誘導ROS產生………………………………………………..………………..5
E. 誘導細胞凋亡……………………………………………..……………………5
蛋白酶體抑制劑MG132…………………………………………………………...6
K562細胞株………………...………………………………………………...…….7
材料與方法……………………………………………………………………………8
藥品與試劑……………………………………………………………..…………..8
實驗方法…………………………………………………………………………10
細胞繼代培養 (Cell culture)…………………………………………………10
細胞週期分析 ( cell cycle analysis )…………………………………………10
細胞死亡分析 ( cell death assay )………………………………………..……11
A. Annexin V/PI stain……………………………………………………….11
B. TUNEL assay…………………………………………………………….11
C. Dye exclusion assay……………………………………………………...12
細胞增生分析 ( cell proliferation assay )……………………………………..13
A. MTT assay………………………………………………………………..13
B. CFSE assay……………………………………………………………….13
RONS 分析…………………………………………………………………….14
粒線體膜電位(Mitochondrial membrane potential ΔΨm )
分析……………………………………………………………………………..15
細胞免疫化學染色分析 ( Immunocyto chemistry staining )…………………15
統計分析 ( Statistical analysis)………………………………………………...16
實驗結果……………………………………………………………………………..17
一、MG132誘導K562細胞死亡之機制………….…………………………………17
MG132對慢性骨髓白血病細胞K562細胞週期之影響…………………..…17
MG132透過caspase-dependent路徑誘導K562細胞凋亡……………………..17
MG132誘導K562細胞內RONS的增加以及破壞粒線體膜電位(Mitochondrial
Membrane Potential, ΔΨm )…………………………………………………….20
AG、NAC以及BAPTA-AM抑制MG132所造成的細胞死亡…………………23
AG以及NAC抑制MG132所誘導K562細胞內RONS的增加、ΔΨm 損傷
以及細胞凋亡………………………………………………………..…………24
MG132所誘導之RONS相關分子…………………………………………….26
二、MG132影響K562細胞週期之變化……………………………………….…...28
MG132藉由誘導K562細胞G2/M phase停滯以及細胞凋亡進而抑制細胞增生
…………………………………………………………………………………..28
AG以及NAC抑制MG132所造成的G2/M phase停滯以及增加MG132所減
少的細胞分裂次數…………………………………..………………………..30討論……………………………………………………….……………………….32
MG132誘導細胞凋亡…………………………………………………………….32

MG132抑制K562細胞增生……………………………………………………..34
蛋白酶體抑制劑可應用在對Glivec具有抗藥性的細胞………………………..35
蛋白酶體抑制劑的抗藥性………………………………………………………..35
結論……………………………………………………………………………..……37
圖目錄………………………………………………………………………………..38
圖一、MG132對K562細胞週期之影響……………………………………..…38
圖二、MG132長時間作用下所誘導之K562細胞死亡…………….…..…..….39
圖三、MG132所誘導之K562細胞凋亡……………………………………..…40
圖四、MG132透過caspase-dependent路徑誘導K562細胞凋亡…….……….41
圖五、MG132誘導K562細胞內RONS增加………………………………42
圖六、NADPH oxidase以及Xanthine oxidase不參與MG132誘導的K562
細胞死亡……………………………………………………………..…....43
圖七、MG132破壞K562細胞粒線體膜電位 (Mitochondrial membrane potential,
ΔΨm)…………………………………………………………….……44
圖八、MG132作用不同時間下K562細胞存活率…………………………45
圖九、AG、NAC以及BAPTA-AM恢復被MG132所抑制的K562細胞存活
率………...……………………………………………………………..46
圖十、eNOS以及nNOS不參與MG132誘導的細K562胞死亡…………….47
圖十一、AG以及NAC抑制MG132所誘導的RONS…………………….48
圖十二、AG以及NAC抑制MG132所誘導的K562細胞ΔΨm的破壞….49
圖十三、AG、NAC以及BAPTA-AM有效抑制MG132所誘導的細胞凋亡……………………………….………………………………………50
圖十四、AG以及NAC抑制MG132所誘導之iNOS、nitrotyrosin、catalase以及恢復MG132所抑制之MnSOD……………………….……………51

圖十五、MG132抑制K562細胞增生…………………………………………….52
圖十六、MG132誘導K562細胞G2/M phase停滯……………………………….53
圖十七、MG132誘導G2/M phase細胞凋亡……………………………………54
圖十八、AG以及NAC抑制MG132所誘導之G2/M phase停滯以及增加細胞分裂次數……………………………………………………….…….…55
表目錄………………………….……………………………………………………56
表一、AG以及NAC分別與1.4 μM MG132作用24小時，ΔΨm受損之細胞比
率與細胞凋亡比率結果比較………………………..………………...56
表二、1.4 μM MG132作用不同時間點，K562細胞週期分布，以及RONS變
化情形……………………..………………………………………………57
表三、AG或NAC與1.4 μM MG132作用24小時，細胞內RONS以及ΔΨm
變化之情形………………………………………………………………..58
附錄…………………………………………………………………………………..59
附錄 圖一、蛋白酶體的結構…………………………………………………...59
附錄 圖二、ubiquitin-proteasome路徑………………………………………….60
附錄 圖三、MG132結構………………………………………………………...61
附錄 表一、尚在臨床試驗中的蛋白酶體抑制劑的作用機制 ……………….62
附錄 表二、蛋白酶體抑制劑利用不同調控機制誘導癌細胞週期停滯………63
附錄 表三、細胞內氧化及抗氧化酵素以及分子………………………………64
參考文獻……………………..………………………………………………………65

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