臺灣博碩士論文加值系統

粒線體生合成(mitochondrial biogenesis)會受到細胞核和粒線體之genome的調控。其中，三個重要的調控因子分別是mitochondrial transcription factor A (TFAM)、nuclear respiratory factors (NRFs)和PPARγ coactivator-1α (PGC-1α)。TFAM是位於粒線體內調控粒線體DNA (mtDNA) 複製和轉錄的相關因子。NRFs和PGC-1α則是在細胞核內的轉錄調控者，此兩個因子會相互作用並活化TFAM，進而調控相關粒線體生合成的基因。過去研究顯示粒線體之功能異常時，細胞核genome和粒線體 genome之間可能會發生cross-talk，而增加粒線體生合成之表現，但是cross-talk的確實機制尚未被研究清楚。有鑑於此，本實驗以不具有粒線體DNA的143B-ρ0細胞，探討氧化磷酸化反應功能的缺陷是否導致粒線體生合成之相關基因的表現變化，以及tricarboxylic acid cycle (TCA cycle) 中重要酵素citrate synthase或呼吸鏈之complex II之活性的變化。除此之外，為了排除去除粒線體DNA的過程中可能造成未知的細胞核DNA變異，而可能造成粒線體生合成之相關基因之表現差異，因此利用重新放回wild-type粒線體DNA的143B-cybrid (cybrid) 細胞作為對照，綜合比較探討143B、143B-ρ0以及cybrid細胞中粒線體生合成之相關基因之變化情形。我們使用西方墨點法來分析粒線體生合成之相關基因之蛋白表現量，使用real time RT-PCR分析PGC-1α之mRNA表現情形，最後測定citrate synthase和complex II之酵素活性。結果顯示，cytochrome c和complex V之ATP synthase β subunit的蛋白質表現量在143B-ρ0細胞中明顯比在143B和cybrid中還高，而complex II之succinate dehydrogenase subunit B和TFAM在143B-ρ0細胞之表現則比在143B和cybrid中為低，然而，complex III之core 1蛋白質在三種細胞中的表現沒有顯著性差異。綜合西方墨點法之結果，因此推測NRFs對於粒線體生合成之相關基因之表現不是主要或是唯一的調控因子。更進一步地，與143B或cybrid比較，143B-ρ0細胞中的PGC-1α之mRNA表現較低，complex II之活性在143B-ρ0細胞中降低，而citrate synthase之酵素活性則較高。因此，在缺乏粒線體DNA之細胞內粒線體生合成之相關基因、蛋白或酵素活性的卻有所變化。

Mitochondrial biogenesis is regulated by the coordinated expression of nuclear genome and mitochondrial genome. Among them, three important transcriptional factors are mitochondrial transcription factor A (TFAM), nuclear respiratory factors (NRFs) and PPARγ coactivator-1α (PGC-1α). TFAM, which is localized in the mitochondria, regulates factors involving the replication and transcription of mtDNA. NRFs and PGC-1α, which are transcriptional factors in the nucleus, can interact and activate TFAM, and subsequently regulate the mitochondrial biogenesis genes. Previous studies shows that mitochondrial dysfunction could lead to cross-talk between nuclear genome and mitochondrial genome and increase the expression of mitochondrial biogenesis, but the exact mechanism of cross-talk has not been studied clearly yet. Based on this, we have used the mtDNA-depleted 143B (143B-ρ0) cell to investigate whether the defect of oxidative phosphorylation (OXPHOS) may result in alterations of expression of mitochondrial biogenesis genes, the activity of citrate synthase in tricarboxylic acid cycle (TCA cycle), and the activity of respiratory complex II. In addition, in order to exclude the possibility that the mtDNA-depletion process which might cause unknown nuclear DNA alterations could result in the changes of expression of mitochondrial biogenesis genes, we utilized the 143B-cybrid (cybrid) which was generated from 143B-ρ0 cell replenished with wild-type mtDNA for comparison to compare the alterations of mitochondrial biogenesis genes in 143B, 143B-ρ0, and cybrid cells. We used Western blot to analyze the protein levels of the genes related to mitochondrial biogenesis, employed real time RT-PCR to analyze mRNA levels of PGC-1α, and finally determined the activities of citrate synthase and complex II. The result showed that the expression of cytochrome c and ATP synthase β subunit of complex V in 143B-ρ0 cell were higher than that in 143B and cybrid cells, while levels of succinate dehydrogenase subunit B of complex II and TFAM were decreased in 143B-ρ0 cells. However, there was no difference in the level of core 1 protein of complex III among three cell lines. Taken together, we speculate that NRFs may not be the major or only factor that control mitochondrial biogenesis genes from results of Western blot analysis. Moreover, the mRNA level of PGC-1α was higher, the activity of complex II was lower, and the activity of citrate synthase was higher in 143B-ρ0 cells compared to 143B or cybrid cells. Therefore, mitochondrial biogenesis-related genes, proteins, or enzyme activities were indeed altered in mtDNA-depleted cells.

指導教授同意書……………………………………..……………………
論文口試委員會審定書…………………………………………..………
長庚大學授權書…………………………………………………..…. iii
誌謝………………………………………………………………….…..iv
中文摘要………………………………………………..……………… vi
英文摘要…………………………………………………………...…...Vii
目錄……………………………………………………………………...ix
第一章 簡介……………………………………………………………..1
1.1 粒線體和粒線體DNA………………………………………...1
1.2 粒線體生合成的調控………………………………………….2
1.3 Mitochondrial dysfunction & up-regulation of mitochondrial biogenesis genes………………………………………………………….7
1.4 143B-ρ0細胞和143B-cybrid細胞……………………………..9
1.5 相關研究之文獻回顧………………………………………...12
第二章 實驗之假說和目的…………………………………………....15
第三章 實驗材料和方法……………………………………………....11
3.1 細胞株和細胞培養…………………………………………...21
3.2 西方墨點轉漬雜交分析所需之細胞蛋白質萃取…………...21
3.3 蛋白質濃度之定量……………………………………….…..22
3.4 西方墨點轉漬雜交分析 (Western blot analysis)…………....22
3.5 細胞RNA之萃取……………………………………………25
3.6 RNA濃度之定量………………………………………….….26
3.7 以Reverse transcription製作cDNA……………………..….27
3.8 PGC-1α基因之Universal ProbeLibrary™ (UPL) Probe之挑選
和偵測引子之設計...... ..............................................................28
3.9即時聚合酶鏈鎖反應 (Real-time PCR)以偵測PGC-1α之mRNA的表現量………………....……………………………29
3.10相對定量方式定量基因之mRNA表現量……………...….30
3.11測定酵素活性所需之細胞蛋白質萃取………………..……32
3.12 Complex II活性分析…………………………………..……32
3.13 Citrate synthase酵素活性分析…………….....….......……...34
3.14統計分析………………………………….…………………35
第四章 實驗結果……………………………..……………………….25
4.1 三種細胞內部，由粒線體DNA製造之呼吸鏈酵素的蛋白質
表現。………………………………………………………….37
4.2 三種細胞內部，NRFs調控細胞核內相關氧化磷酸化反應的
蛋白質表現。………………………………………………….37
4.3 三種細胞內部，PGC-1α之mRNA表現。……………......38
4.4 三種細胞之粒線體內complex II和citrate synthase的酵素活
性比較。………………………………………………………40
第五章 結果與討論…………………………………………………..41
參考文獻………………………………………………………………50
Figures目錄
Figures 1. 在三個細胞中，由粒線體DNA載錄之COX II之蛋白質
表現……………………………………………………………60
Figures 2. 在三種細胞中，complex II之succinate dehydrogenase
subunit B的蛋白質表現………………………………….……61
Figures 3. 在三個細胞中，complex III之core 1 protein的表現.62
Figures 4. 在三個細胞中，Complex V之ATP synthase β subunit
的蛋白質表現………………………………………………….63
Figures 5. 在三個細胞中，cytochrome c的蛋白質表現………..64
Figures 6. 在三個細胞中，TFAM的蛋白質表現……….………65
Figures 7. RNA電泳圖……………………………………..……..66
Figures 8. PGC-1α之calibration curve建立………………..……67
Figures 9. β-actin之calibration curve建立………………………69
Figures 10. 三種細胞內部，PGC-1α之mRNA表現量...............71
Figures 11. 三種細胞之粒線體內呼吸鏈上，complex II之活….72
Figures 12. 三種細胞之三羧酸循環上citrate synthase之活性…73
Table目錄
Table 1. 本實驗中，受到NRFs調控細胞核內相關氧化磷酸化反應
之蛋白質的表現情況之總整理……………..………………...74
Table 2. 與相關研究之文獻的結果比較………………………....75
Table 3. 本實驗中，complex II和citrate synthase的酵素活性和蛋
白質的表現情況之整理………………..………………………76
附圖目錄
附圖一、呼吸酵素複合體內，由細胞核DNA和粒線體DNA分別
合成的polypeptides以及分布位置…………………………….77
附圖二、粒線體的生合成和功能性主要依賴許多細胞核之genome
的調控表現……………………………..……………………....78
附圖三、NRF-1辨識於cytochrome c promoter的位置…..……..80
附圖四、NRF-1和NRF-2辨識於TFAM promoter的位置……..81
附圖五、PGC-1α調控粒線體生合成的途徑…………………..…82
附圖六、Transcriptional activators和coactivators調控粒線體的
function和biogenesis的關係圖………………………………..83
附圖七、transmitochondrial cell lines的製造原理………….…….84
附圖八、PGC-1α之UPL probe和引子資料...................................86
附圖九、β-actin之UPL probe和引子資料.....................................87
附表目錄
附表一. NRFs調控細胞核內相關氧化磷酸化反應之蛋白質…….88

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