探討MEHP對肌肉粒線體代謝功能的影響

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陳怡嬛(I-Huan Chen) 查詢紙本館藏

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生命科學系

論文名稱

探討MEHP對肌肉粒線體代謝功能的影響
(The influence of MEHP on metabolic and mitochondrial functions in skeletal muscle)

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摘要(中)

DEHP為最常用於塑料的化合物，與人們的生活息息相關，暴露在醫療器材、食品包裝、建築材料、化妝品、玩具等。DEHP一旦經由各種途徑吸收，就會快速代謝成MEHP。之前的研究指出，塑化劑可能提高胰島素阻抗及第二型糖尿病的風險，而骨骼肌又與代謝症候群有關，因此我們以C2C12為研究題材，觀察C2C12在處理100μM MEHP後對分化、代謝基因表現量及粒線體功能的影響。在肌肉生成(myogenesis)過程中，細胞增殖狀態下給予MEHP會抑制分化，而分化4天後給予MEHP抑制分化的效果會顯著降低，因此以分化3天後再給予MEHP作為後續實驗，避免代謝作用受分化影響。葡萄糖攝取實驗顯示MEHP對細胞因受insulin刺激而增加的葡萄糖攝取並沒有影響，而在醣類與脂肪酸代謝方面，ERRα、PDK4和CPT1B基因的表現量皆有顯著上升。透過穩定細胞株C2C12-MTS-RFP觀察粒線體細胞形態，在CMB時期給予2天MEHP其粒線體趨向分裂狀態，而透過測定粒線體的DNA含量，發現不論在哪一個時期給予MEHP皆無影響，此外Tfam基因的表現量也沒有差異。為了得知粒線體功能是否受到影響，我們測定一系列在電子傳遞鏈過程中會發生的反應。給予MEHP不會影響到肌管時期的膜電位但是會讓succinate dehydrogenase活性下降，並且使ROS含量與SOD活性些許上升，而HO-1基因表現量也有所上升。接著我們測定電子傳遞鏈上各個具代表性的complex subunit 蛋白質的表現量，發現MEHP會影響到complex I subunit-Ndufb8的含量。為了確認MEHP影響Ndufb8的轉錄或轉譯，以Real time PCR及promoter activity 測定結果皆沒有受到MEHP影響。透過給予轉譯抑制劑cycloheximide 顯示MEHP可能影響Ndufb8的轉譯。綜合以上所述，塑化劑可能造成粒線體功能失調，並減少insulin的敏感度。

摘要(英)

Di(2-ethylhexyl) phthalate (DEHP) is widely used in industry to increase the malleability of polyvinyl chloride. It can be found in plastic package, toys, clinic, cosmetic, building, and so on. Once entered the body, it is quickly metabolized to MEHP. Previous studies have shown that the level of phthalate is associated with the development of type-2 diabetes and insulin resistance. In this study, the effects of MEHP on myogenic differentiation, the metabolism of glucose and fatty acid oxidation, and mitochondrial function were observed. We treated C2C12 cells with 100 μM MEHP for two days and cells treated with MEHP in the proliferation stage during myogenesis failed to form myotubes. After three days of differentiation, MEHP treatment did not influence differentiation, we used this stage for measuring the effects of MEHP on metabolism so the intereference from differentiation could be avoided. First, we measured glucose uptake, in the stimulation of insulin situation, MEHP group caused the cells to absorb less glucose compared to the DMSO group. Moreover, we investigated the metabolism of glucose and fatty acid oxidation by using Real-time PCR. The gene expression of ERRα、PDK4、CPT1B is up-regulated. Second, we observed the effect of MEHP on mitochondrial function. C2C12-MTS-RFP was culture for three days in CMB stage, then treated with MEHP for two days and checked the mitochondrial morphology. The cells tended to become fission as compared to the DMSO group. It probably had poor function in mitochondria. So we checked the content of mitochondria. MITO DNA and the gene expression of Tfam was not affected by MEHP. To investigate the function of mitochondria, a series assays on the functions of electron transport system was measured. Membrane potential was not influenced by MEHP, while succinate dehydrogenase was decreased and produced ROS then enhance SOD activity. The expression of ROS-related gene HO-1 was significantly up-regulated. Third, we measured the protein level of 5 complexes in the electron transport system. In the myotube stage, treatment with MEHP decreased the lelvel of complex I subunit-Ndufb8. To ensure whether MEHP influenced transcription or translation stage, mRNA level and promoter activity were measured, and we found MEHP did not influence the transcription of Ndufb8. Through adding translation inhibitor cycloheximide in the medium, we found MEHP might interfere with the translation of Ndufb8. Taken together, MEHP can affect the function of mitochondria and by affecting the functions/levels of key factors in the ETC system.

關鍵字(中)

★ 塑化劑
★ 肌肉
★ 粒線體

關鍵字(英)

論文目次

中文摘要 I
Abstract II
致謝 IV
聲明 (Declaration) V
縮寫表 VI
第一章緒論 - 1 -
1.塑化劑對動物及人體代謝的影響 - 1 -
2. 肌肉分化 (Myogenesis) - 4 -
3.粒線體在肌肉分化中的角色 - 5 -
3-1粒線體基因 - 5 -
3-2粒線體的生合成 - 5 -
3-3粒線體的功能 - 6 -
4. 研究動機與目的 - 8 -
第二章實驗材料與方法 - 9 -
2-1 實驗材料 - 9 -
2-1-1. 細胞株 - 9 -
2-1-2. 細胞培養 - 9 -
2-2. MTT assay - 9 -
2-3. Immunofluorescence - 9 -
2-4. 種C2C12-MTS-RFP玻片 - 10 -
2-5. 抽取gDNA - 10 -
2-6. 膜電位測定 - 11 -
2-7. ROS 測定 - 12 -
2-8. SOD測定 - 12 -
2-9. SDH activity - 13 -
2-10. 抽RNA - 14 -
2-10-1. Reverse Transcription (翻轉成cDNA) - 15 -
2-10-2. Real time PCR - 15 -
2-11. glucose uptake - 16 -
2-12. 西方墨點轉漬法(western blot) - 17 -
2-13.質體建構 - 18 -
2-14. Transient transfection - 21 -
2-15. Luciferase reporter assay - 22 -
2-16.建立TRE-EGFP Ndufb8 - 22 -
2-17. 建立穩定細胞株 - 23 -
第三章實驗結果 - 24 -
Ⅰ. 選取100µM MEHP作為實驗濃度 - 24 -
II. 在不同階段處理MEHP 觀察分化狀態，分化3天後處理MEHP 2天，肌管的形成有下降趨勢但不顯著 - 24 -
III. MEHP皆不會影響C2C12 mtDNA的含量 - 25 -
IV. MEHP影響C2C12 MT時期的ROS、SOD及SDH活性 - 25 -
V. MEHP促使粒線體形態趨向分裂(Fission) - 26 -
VI. MEHP對細胞代謝相關基因的影響 - 27 -
VII. MEHP對於葡萄糖攝取的影響 - 27 -
ⅥII. MEHP對於粒線體Complex 蛋白質影響 - 28 -
IX. MEHP調控Ndufb8的轉譯 - 28 -
X. 在C2C12細胞中大量表現Ndufb8 - 30 -
第四章討論 - 31 -
Ⅰ. MEHP對肌肉細胞分化的影響 - 31 -
II. MEHP影響粒線體功能 - 32 -
Ⅲ. MEHP使C2C12相對於DMSO葡萄糖攝取量減少 - 34 -
Ⅳ、結論 - 35 -
第五章實驗結果圖 - 36 -
Fig. 5-1 不同濃度MEHP對C2C12於CMB及MT時期細胞存活率結果 - 37 -
Fig. 5-2 MEHP對C2C12肌管時期型態的影響 - 38 -
Fig. 5-3 C2C12在不同時期處理MEHP對分化狀況的影響 - 39 -
Fig. 5-4 C2C12在不同培養階段處理MEHP對粒線體DNA含量的影響 - 40 -
Fig. 5-5 MEHP對C2C12粒線體膜電位的影響 - 41 -
Fig. 5-6 MEHP對C2C12分化時期粒線體功能的影響 - 42 -
Fig. 5-7 以C2C12-RFP-MTS 細胞株觀察粒線體細胞型態 - 43 -
Fig. 5-8 MEHP對C2C12分化過程基因表現的影響 - 44 -
Fig. 5-9 MEHP對於C2C12 CMB及MT時期給予insulin後葡萄糖攝取的影響 - 45 -
Fig. 5-10 MEHP對C2C12細胞CMB及MT時期蛋白質表現量的影響 - 47 -
Fig. 5-11 MEHP對PDK4, CPT1B, NDUFB8 promoter活性的影響 - 48 -
Fig. 5-12 MEHP及cycloheximide對C2C12分化的影響 - 52 -
Fig. 5-13 MEHP及cycloheximide對C2C12分化的影響 - 54 -
Fig. 5-14 建立TRE-EGFP-Ndufb8 穩定細胞株 - 55 -
Fig. 5-15 Overexpression of Ndufb8 in C2C12 - 56 -
第六章參考文獻 - 57 -
附錄 - 60 -
附錄一: RT-PCR Primers - 60 -
附錄二: PCR primer-for gDNA - 62 -
附錄三:質體 primers - 62 -
附圖一:TRE-EGFP-NDUFB8 CDS Sequence結果 - 62 -
附圖二: Pstable-NDUFB8 promoter Sequence結果 - 63 -
附錄四: 溶液配方 - 64 -

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2019-7-30

推文