過氧化氫體於肌肉形成過程及癌症惡質症的調控與角色

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吳傳喆(Chuan-Che Wu) 查詢紙本館藏

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論文名稱

過氧化氫體於肌肉形成過程及癌症惡質症的調控與角色
(The Regulation and Role of Peroxisome in Myogenesis and Cancer Cachexia)

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摘要(中)

過氧化氫體(Peroxisome)為細胞代謝之重要胞器，其功能包括協助細胞進行呼吸作用、長碳鏈脂肪酸代謝、活性氧類(ROS)代謝等。細胞中過氧化氫體生合成蛋白(Peroxisomal biogenesis factor, Peroxins)負責過氧化氫體的增生及發育，主要的增生方式分成由 Pex3, Pex16, Pex19 為主的過氧化氫體新生合成(de novo synthesis)以及由 Pex11ß 為主的過氧化氫體分裂增生(Peroxisome fission) 這兩種方法。在實驗室先前的研究中發現，隨著肌肉生成過程，過氧化氫體的數量也會逐漸增加，暗示了肌肉生成過程與過氧化氫體之間的相互關係；而在癌症惡質症環境中，過氧化氫體相關基因也呈現下降的趨勢。因此，本篇便著重探討過氧化氫體於肌肉生成及癌症惡質症之間的關聯性。
實驗中我們發現，在肌肉生成過程中，不論是 in vivo 或 in vitro，過氧化氫體相關基因的表現皆呈現逐漸上升的趨勢，且過氧化氫體的新生數量也逐漸增加。同時觀察到，MRFs(Myogenic regulatory factor)會直接對 Pex3, Pex16, Pex19 啟動子進行調控。而上述實驗結果指出，Pex3 在肌肉生成過程中似乎扮演著很重要的角色。在藉由 knockdown Pex3 的細胞株中觀察到，大部分大量表現 Pex3 細胞株中觀察到除了過氧化氫體數量及形態上的改變，過氧化氫體及粒線體的功能並沒有太大的改變，然而在大量表現 Pex3 後細胞的分化效率卻有顯著地下降，指出在肌肉中，Pex3 對於過氧化氫體及粒線體扮演著舉足輕重的角色，並且在肌肉分化過程需要被精準地調控。此外，在經過有氧呼吸運動訓練後發現，快縮肌與慢縮肌之間過氧化氫體新生合成因子表現方式不同，並且觀察到在肌肉中過氧
化氫酶並不位於過氧化氫體內。
在早發性癌症惡病質小鼠內，觀察到在快縮肌內 Pex16 及 Acox1 異常上升，同時藉由 RNAseq 分析發現在晚發性小鼠快縮肌可以觀察到過氧化氫體相關基因大量被影響。此外，早發性與晚發性小鼠之間，大部分的 MRFs 表現趨勢類似，唯獨 MyoG 在早發性小鼠中出現異常上升，並且在快縮肌組別中出現較嚴重的症狀。將大量表現 MyoG 細胞株培養於癌症惡質症的環境中觀察到，兩者的加成性會影響肌肉分化並導致肌肉萎縮，造成癌症惡質症的惡化。

摘要(英)

Peroxisome is an important organelle for cell metabolism, including reducing ROS(Reactive oxygen species), very long-chain fatty acid, lipid metabolism, and so on.Peroxisomal biogenesis factors (Peroxin) are involved in several procedures of peroxisome biogenesis, and in the two major ways regulating the number of peroxisomes, de novo synthesis is regulated by Pex3, Pex16, and Pex19 while growth and division is mainly regulated by Pex11β. As the number of peroxisomes has been found to be increased during myogenesis in our previous study, therefore, in this thesis, I want to clarify how the peroxisome is regulated in myogenesis and cancer cachexia.
Here I found the expression of many peroxisome related genes is increased during myogenesis both in vitro and in vivo. Meanwhile, MRFs (Myogenic regulatary factors) show their ability to regulate Pex3, Pex16, Pex19 promoters derectly. These data all points out that Pex3 plays an important role in myogenesis. In Pex3 knockdowned cells, neither peroxisome nor mitochondria was functioning properly, and the number and morphology of both organelles were changed significantly. Furthermore, less total and tubular shape peroxisomes were observed in these cells. On the other hand, in Pex3 over-expressed cells, the number of peroxisome was increased, and more tubular shape peroxisomes were found. However, the function of peroxisome and mitochondria were
both the same as that in controll cells. However, over-expression of Pex3 lead to poorer myogenesis, indicating that Pex3 level is critically regulated during myogenesis.
Interestingly, after aerobic exercise training, peroxisome related genes were differentially regulated in different muscle types, the expression level of Pgc1α is similar to Pex19 level but not Pex3. We were also surprised to find the location of Catalase out of peroxisome in myofibers in vivo.
In Cancer cachexia, the expression of Pex16 and Acox1 was abnormally increased in fast-twitch SKM. With RNAseq analysis, the peroxisome related genes were found
significantly regulated in fast-twitch SKM of late onset mice. Meanwhile, MRFs were mostly the same in both early onset and late onset groups, but only MyoG was abnormally absent in fast-twitch SKM of early onset group. In MyoG over-expressed cells, myogenesis was functionally normal, but serious muscle atrophy and myogenesis dysfunction were observed when MyoG and cachexia signals were combined.

關鍵字(中)

★ 過氧化氫體
★ 肌肉生成過程
★ 粒線體
★ 癌症惡質症

關鍵字(英)

★ Peroxisome
★ Myogenesis
★ Mitochondria
★ Cancer cachexia

論文目次

目錄
摘要(Abstract)------------------------------------------------------------------------------------I
聲明(Declaration)------------------------------------------------------------------------------III
致謝(Acknowledgment)-----------------------------------------------------------------------IV
目錄(Content)------------------------------------------------------------------------------------V
縮寫與全名對照表(Abbreviations)----------------------------------------------------------X
第一章、緒論
1-1. 過氧化氫體(General introduction of Peroxisome)------------------------------1
1-2. 粒線體(General introduction of mitochondrion)--------------------------------1
1-3. 過氧化氫體與粒線體(Peroxisome-Mitochondria connection)---------------2
1-4. 過氧化氫體生合成因子(Peroxisome biogenesis factors)---------------------2
1-4-1.過氧化氫體基質蛋白運輸(Peroxisomal matrix protein import)-------3
1-4-2.過氧化氫體膜生合成(Peroxisomal membrane protein biogenesis)----3
1-4-3.過氧化氫體分裂增生(Peroxisomal fission proliferation)---------------4
1-5. 肌肉生成過程(The processes of myogenesis)-----------------------------------5
1-6. 過氧化氫體與肌肉生成關係(Relation between Pex and myogenesis)------5
1-7. 癌症惡質症(cancer cachexia)-----------------------------------------------------6
1-8. 研究動機與研究問題(Research motivation and specific aims)--------------6
第二章、材料及方法
2-1. 實驗材料(Experiment materials)--------------------------------------------------9
2-2. 質體建構(Plasmid establishment)-----------------------------------------------10
VI
2-3. 細胞轉染(Transfection)-----------------------------------------------------------13
2-4. 冷光素酶報導檢測(Luciferase reporter assay)--------------------------------14
2-5. 活體細胞過氧化氫體追蹤法(Peroxisome cellulo pulse chase assay)------14
2-6. RNA 萃取(RNA extraction)------------------------------------------------------15
2-7. 反轉錄聚合酶連鎖反應(Reverse-transcription PCR, RT-PCR)------------15
2-8. 即時定量聚合酶連鎖反應(Quantitative real-time PCR, qRT-PCR)-------16
2-9. 細胞免疫螢光染色(Immunofluorescence, IF)---------------------------------16
2-10. 小鼠組織切取(Mouse tissue extraction)---------------------------------------17
2-11. 小鼠低強度有氧運動(Low-intensity oxidative exercise)--------------------17
2-12. 組織冷凍切片(Frozen section)---------------------------------------------------18
2-13. CRISPR/Cas9 小型引導 RNA 設計(CRISPR/Cas9 sgRNA design)-------18
2-14. CRISPR/Cas9 慢病毒製造(CRISPR/Cas9 lentiviral production)-----------19
2-15. CRISPR/Cas9 慢病毒轉染(CRISPR/Cas9 lentiviral transfection)----------19
2-16. 西方墨點法(Western blot)--------------------------------------------------------20
2-17. 脂肪酸代謝測定(Measurement of Fatty acid β-oxidation )------------------21
2-18. 粒線體膜電位差檢測(MITO Membrane potential Δψm)--------------------22
2-19. 活性氧物質含量檢測(Measurement of ROS level)---------------------------23
2-20. NAD(P)H 和 FAD 含量檢測(NAD(P)H and FAD level)--------------------24
2-21. 小鼠肌肉星狀細胞抽取(Mouse Satellite cell extraction)--------------------25
2-22. 小鼠癌症惡質症模型實驗(Mouse Cachexia experiment)-------------------25
2-23. 統計方法----------------------------------------------------------------------------26
VII
第三章、結果
3-1. 肌肉發育過程中過氧化氫體生合成及相關基因表現----------------------27
3-2. 肌肉發育過程中過氧化氫體及粒線體相關蛋白表現量-------------------27
3-3. 肌肉發育過程中過氧化氫體新生數量及效率-------------------------------28
3-4. MRFs 對 Pex3, Pex16, Pex19 啟動子的調控---------------------------------29
3-5. C2C12-shPex3 穩定細胞株 PEXO 及 MITO 相關基因表現---------------30
3-6. C2C12-shPex3 過氧化氫體數量------------------------------------------------31
3-7. C2C12-shPex3 粒線體形態及細胞相關代謝功能--------------------------31
3-8. C2C12-tTA-hPex3 穩定細胞株 Pexo 及 Mito 相關基因表現--------------32
3-9. C2C12-tTA-hPex3 肌肉生成過程及基因表現--------------------------------33
3-10. C2C12-tTA-hPex3 過氧化氫體數量及形態變化-----------------------------34
3-11. C2C12-tTA-hPex3 粒線體形態及細胞相關代謝功能-----------------------34
3-12. 建立 C2C12-dCas9-KRAB-sgPex3 穩定細胞株------------------------------35
3-13. 小鼠有氧運動過氧化氫體基因表現變化-------------------------------------36
3-14. 小鼠有氧運動對肌肉種類及過氧化氫體分佈影響-------------------------36
3-15. 胚胎發育過程肌肉生成及過氧化氫體分佈----------------------------------36
3-16. 癌症惡質症小鼠基因表現變化-------------------------------------------------37
3-17. 癌症惡質症小鼠早發性及晚發性基因表現----------------------------------38
3-18. 癌症惡質症小鼠早發性及晚發性基因大數據分析-------------------------38
3-19. 癌症惡質症對分化後 C2C12-tTA-MyoG 的影響----------------------------39
第四章、討論
4-1. 肌肉生成過程中過氧化氫體及相關基因的調控----------------------------40
VIII
4-2. 減弱 Pex3 表現量對肌纖維母細胞造成的影響------------------------------41
4-3. 大量表現 Pex3 對肌肉分化造成的影響---------------------------------------42
4-4. 運動對於過氧化氫體生合成因子基因表現造成的影響-------------------43
4-5. 癌症惡質症對過氧化氫體及肌肉萎縮的影響-------------------------------43
4-6. 結論及未來方向-------------------------------------------------------------------44
第五章、圖表
5-1. 肌肉發育過程中過氧化氫體生合成及相關基因表現----------------------45
5-2. 肌肉發育過程中過氧化氫體及粒線體相關蛋白表現量-------------------46
5-3. 肌肉發育過程中過氧化氫體新生數量及效率-------------------------------47
5-4. MRFs 對 Pex3, Pex16, Pex19 啟動子的調控---------------------------------48
5-5. C2C12-shPex3 穩定細胞株 PEXO 及 MITO 相關基因表現---------------49
5-6. C2C12-shPex3 過氧化氫體數量及形態變化---------------------------------50
5-7. C2C12-shPex3 過氧化氫體型態改變------------------------------------------51
5-8. C2C12-shPex3 粒線體形態及細胞相關代謝功能--------------------------52
5-9. C2C12-hPex3-tTA 穩定細胞株 Pexo 及 Mito 相關基因表現--------------53
5-10. C2C12-hPex3-tTA 肌肉生成過程及基因表現--------------------------------54
5-11. C2C12-hPex3-tTA 過氧化氫體數量及形態變化-----------------------------55
5-12. C2C12-hPex3-tTA 粒線體形態及細胞相關代謝功能-----------------------56
5-13. 建立 C2C12-dCas9-KRAB-sgPex3 穩定細胞株------------------------------57
5-14. 小鼠有氧運動過氧化氫體基因表現變化-------------------------------------58
5-15. 小鼠有氧運動對肌肉種類及過氧化氫體分佈的影響----------------------59
5-16. 胚胎發育過程肌肉生成及過氧化氫體分佈----------------------------------60
IX
5-17. 癌症惡質症小鼠基因表現變化-------------------------------------------------61
5-18. 癌症惡質症小鼠早發性及晚發性基因表現----------------------------------62
5-19. 癌症惡質症小鼠早發性及晚發性基因大數據分析-------------------------63
5-20. 癌症惡質症對分化後 C2C12-tTA-MyoG 的影響----------------------------64
第六章、參考文獻
6-1. Reference----------------------------------------------------------------------------65
第七章、附錄
7-1. 溶劑及溶液配方(The protocol of solvent and solution protocol)-----------69
7-2. 藥品試劑----------------------------------------------------------------------------71
7-3. 抗體(Antibody)---------------------------------------------------------------------71
7-4. 引子條目(Primer list)--------------------------------------------------------------72
7-5. 補充圖表(Suppementary data)---------------------------------------------------75
7-6. RNAseq------------------------------------------------------------------------------77

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2022-8-8

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