臺灣博碩士論文加值系統

丙烯醛 (Acrolein)是具有高度活性的不飽和醛類且普遍存在環境中，例如香菸、汽機車排放煙等等。而過去文獻提到這些環境汙染源和肺部疾病例如慢性肺阻塞疾病以及肺癌相關。根據之前的研究得知acrolein會造成DNA突變以及抑制DNA修復，這些傷害都被發現是導致肺癌的原因之一。過去文獻提出acrolein會導致粒線體的功能喪失，粒線體的功能損害也可能是acrolein造成的肺部疾病的原因之一。由於粒線體擔任細胞能量來源的重要胞器，因此維持粒線體品質就相對重要，粒線體會透過粒線體生成、融合/分裂 (fusion/ fission)以及自噬 (mitophagy)達到維持粒線體品質穩定的目的。當粒線體遇到傷害時會將受損的粒線體分離出來再經由粒線體自噬清清除受損粒線體。然而，對於遇到acrolein造成的氧化性傷害時粒線體動態平衡的運作方式目前還尚未清楚。根據研究結果得知acrolein會引起細胞內以及粒線體內的氧化壓力上升、降低粒線體的膜電位、降低ATP 含量、粒線體DNA拷貝數和粒線體膜蛋白基因的表現量減少。Acrolein對粒線體造成形態趨於分裂，分裂出來的粒線體會透過PINK1穩定以及LC3II導致粒線體自噬。由以上結果得知，acrolein造成粒線體的氧化性壓力會促使粒線體進行分裂和自噬，達到去除受損粒腺體和保護細胞存活的目的。然而若上述方法無法達到修復粒線體功能則誘發細胞凋亡。本次的實驗結果希望能藉由發現粒線體動態平衡以及細胞自噬和粒線體自噬在acrolein毒性扮演的角色能作為探討治療肺部疾病的方向之一。

Acrolein, a ubiquitous environmental pollutant, can be found in cigarette smoke, car exhausts, and overheated cooking oils, which are known as risk factors of lung diseases including asthma, chronic obstructive pulmonary disease (COPD) and lung cancer. Our previous studies have been shown that acrolein induces mutagenic DNA adducts and inhibits DNA repair, which plays an important role in lung carcinogenesis. Mitochondrial homoeostasis is crucial through mitochondrial fission/ fusion cycle, biogenesis, and mitophagy, a selective mitochondrial autophagy. It is not clear that the effect of acrolein on mitochondrial quality control and whether mitochondrial dynamics and mitophagy pathway are involved in acrolein–induced mitochondrial dysfunction. In the present study, our results show that acrolein induces mitochondrial oxidative stress, alteration of mtDNA copy number, and mitochondrial fission which results in mitochondrial dysfunction. Furthermore, mitophagy pathway is induced by acrolein by PINK1 stabilization on mitochondrial membrane and LC3 cleavage. These results suggest that acrolein-induced oxidative stress in mitochondria triggers mitochondrial fission and mitophagy to remove damaged mitochondria as a pro-survival role. However, if a substantial proportion of mitochondrial are damaged, apoptosis ensues. This research may help understand the mechanism of acrolein-induced mitochondrial dysfunction and provide the insight for prevention of acrolein-induced lung diseases.

致謝 ……………………………………………………………………i
中文摘要 ……………………………………………………………………ii
Abstract……………………………………………………………………iii
縮寫檢索表…………………………………………………………………iv
目錄 ……………………………………………………………………vi
目錄圖次 ……………………………………………………………………x
附錄次 ……………………………………………………………………xi
第一章 研究背景 1
1 丙烯醛 Acrolein介紹 1
1-1 Acrolein的來源 1
1-2 Acrolein的代謝 2
1-3 Acrolein的相關疾病 2
2 Acrolein的氧化性傷害 4
2-1 核酸、蛋白質、脂質 4
2-2 細胞內胞器 5
2-3 Acrolein引發的細胞凋亡 7
3 粒線體介紹 8
3-1 粒線體功能 8
3-2 粒線體氧化壓力 8
3-3 粒線體融合/分裂 (Fusion/ fission cycle) 9
3-4 粒線體生成 11
3-5 細胞自噬 (Autophagy)與粒線體自噬 (Mitophagy) 11
3-6 粒線體功能喪失與疾病的關係 13
4 細胞自噬 (Autophgy)與粒線體自噬 (Mitophagy)的疾病的關聯 13
4-1 細胞自噬 (Autophgy) 13
4-2 粒線體自噬 (Mitophagy) 14
第二章 研究目的 15
第三章 研究材料與方法 16
1 實驗材料 16
1-1 藥品與試劑 16
1-2 一級抗體 18
1-3 二級抗體 19
1-4 實驗儀器 20
2 實驗方法 22
2-1 細胞培養 22
2-2 Acrolein 處理 22
2-3 MTT 還原測定 (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay, (MTT) assay) 23
2-4 乳酸脫氫酶活性試驗 (Lactate dehydrogenase release assay, (LDH) release assay) 23
2-5 粒線體膜電位 (JC-1 assay) 24
2-6 細胞活性氧自由基測定（2'-7'-Dichlorodihydrofluorescein diacetate assay, (DCF-DA) assay） 25
2-7 粒線體內的自由基測定 (MitoSOX assay) 26
2-8 細胞內ATP含量之測定 (ATP content assay) 26
2-9 DNA之萃取及定量 27
2-10 細胞total RNA 萃取 28
2-11 即時定量聚合酵素鏈反應 (Real-PCR) 28
2-12 粒線體DNA拷貝數( Mitochondrial DNA copy number)之測定 …………………………………………………………………30
2-13 西方墨點法 (Western analysis) 31
2-14 免疫螢光染色 (Immunofluorescence staining analysis) 32
2-15 粒線體分裂 (mitochondrial fragmentation)定量 33
2-16 細胞週期測定 (cell cycle analysis) 33
2-17 細胞凋亡分析(Anexin-V/ PI analysis) 34
2-18 數據統計分析 (Statisitics analysis) 34
第四章 研究結果 35
1-1 Acrolein對A549以及MRC5的細胞毒殺性 35
1-2 Acrolein對粒線體造氧化性傷害以及功能喪失 35
1-3 Acrolein對於粒線體形態變化的影響 38
1-4 Acrolein對於細胞自噬 (autophagy)粒線體自噬 (mitophagy)的影響 …………………………………………………………………40
1-5 Autophagy在acrolein造成細胞毒性下所扮演的角色 42
1-6 Acrolein對於細胞毒殺性造成死亡的方式 42
第五章 討論 …………………………………………………………………….44
第六章 結論 …………………………………………………………………….48
第七章 參考文獻 49
圖示 …………………………………………………………………….55
附錄 …………………………………………………………………….72

目錄圖次
Fig.1 Acrolein 對於A549 以及MRC5的細胞毒殺性 55
Fig.2 Acrolein 在A549以及MRC5細胞株中引發細胞內與粒線體內ROS過度產生 56
Fig.3 Acrolein 在A549以及MRC5細胞株中對於粒線體DNA copy number 以及粒線體基因的影響 57
Fig.4 Acrolein在A549以及MRC5細胞株中造成粒線體膜電位下降以及ATP含量減少 59
Fig.5 Acrolein 在A549以及MRC5細胞株中對於粒線體形態變化的影響 61
Fig.6 Acrolein 在A549以及MRC5細胞株中促進粒線體分裂 (fission) 63
Fig. 7 Acrolein 在A549以及MRC5細胞株中引發autophagy 65
Fig.8 Acrolein 在A549以及MRC5細胞株中引發mitophagy 68
Fig. 9 Autophagy在A549以及MRC5細胞株中acrolein細胞毒性中所扮演的角色 69
Fig.10 Acrolein在A549以及MRC5細胞株中引發細胞凋亡 71

附錄次
附錄一 Acrolein 代謝 72
附錄二 Fusion/fussion cycle 和p-Drp1 73
附錄三 SDS-PAGE 膠體配置 74
附錄四 西方點墨法緩衝液配置 74

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