博碩士論文 104821011 詳細資訊




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姓名 陳書典(Shu-Dian-Chen)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討塑化劑 DEHP/MEHP 對肌肉代謝基因表現的影響
(Exploring the influence of plasticizer DEHP/MEHP on metabolic gene expression in skeletal muscle)
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摘要(中) 塑化劑是一個廣泛應用於塑膠製品的人工合成物質,適當的塑化劑可以改變塑膠物質的質
地,有便於業者對產品包裝的選擇以利消費者使用,常見的添加物為 di(2-ethylhexyl)
phthalate (DEHP),塑化劑不但是一種化學物質還是一個環境荷爾蒙,進入人體後會轉變
為 mono-(2-ethylhexyl) phthalate (MEHP) 進行代謝,過量的塑化劑進入人體內會造成肝、
腎、前列腺等等的癌症生成,以及海綿體的異常。過去發現 DEHP 會去影響 PPAR 的路
徑,DEHP / MEHP 可以透過活化 PPARs 的細胞核受體來促進過氧化氫體的增生,促使許
多代謝途徑像是脂肪酸代謝、過氧化氫分解及膽固醇生合成等也因此被促進。此外 DEHP
還會與 TRPV1 這個受體鈣離子通道做結合,目前的研究主要在探討 100 μM MEHP 對於
C2C12 肌肉細胞的分化後其代謝基因的影響,以及粒線體 fission and fusion 和過氧化氫體
數量的影響,並透過管餵 DEHP (200 mg/kg/14 days) 於 TRPV1-/-並檢測對 gastrocnemius 代
謝基因的影響。我們發現肌細胞分化後處於 MEHP (100 μM)的環境下肌管變細長之外,
且 PDK4 基因表現量上升了,則表示細胞減少了對於葡萄糖的使用率,而過氧化氫體的數
量沒有明顯變化,但粒線體 fission 比例上升了,然而 fission 的比例增加表明為了維持原有
的 ATP 的量並使細胞避免走向凋亡或者是避免突變的發生所形成的一個保護機制,而在管
餵 DEHP 後的 TRPV1-/-其 gastrocnemius 在 mtDNA/ncDNA level 的量則是下降的。而 RNA
level 的結果可以看到 TRPV1-/-的 PDK4 下降了,表明塑化劑確實有透過 TRPV1 channel 來
影響身體代謝的方式,並對於肌肉有明顯的改變養分選擇的偏好,另外在心臟採血後血清
測量中的血糖、三酸甘油酯、膽固醇皆沒有明顯差異,然而詳細的機制還未釐清,透過了
解塑化劑對於肌細胞的粒線體以及過氧化氫體的影響,並檢測 TRPV1-/-肌細胞的代謝基因
的影響有助於對代謝症候群患者的預防與治療。
摘要(英) Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer for most PVC based medical devices and the
addition of DEHP can increase the flexibility of plastic products. Many of these plasticizers act as
endocrine disrupters and have been linked to the rise of metabolic disorders. Phthalates have been
demonstrated to bind to peroxisome proliferator-activated receptors (PPARs); however, recent
evidences also suggest the involvement of multiple non-PPARα pathways in DEHP-induced
carcinogenesis. Transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) in
skeletal muscle (SKM) is mainly located in the sarcoplasmic reticulum (SR) responding for load
signal induced Ca+2 release and contraction, and it is of interest to test whether DEHP can regulate
its channel activity and result in metabolic changes in SKM. In the present study, C2C12
myotubes was treated with MEHP (100 μM) for 4 days in vitro and mice were treated with DEHP
(200 mg/kg) for 14 days in vivo to observe the effects of DEHP/MEHP on SKM metabolism. We
found that DEHP/MEHP could change SKM metabolism and the expression of some metabolic
genes, such as PDK4, was markedly up-regulated after treatment with DEHP/MEHP. But the
same phenomenon was not observed in TRPV1-/- mice, in which PDK4 gene is down-regulated,
suggesting that a switch of nutrient utilization has occurred. Furthermore, the mitochondrial
content in cells was examined and a down regulation by DEHP was discovered in TRPV-/-
but not
in wildtype mice. In addition, treatment of C2C12 myoblasts with MEHP (100μM) changed
mitochondrial morphology implying functional alteration. Our results support the notion that
DEHP/MEHP is a strong modulator of metabolism in SKM.
關鍵字(中) ★ 塑化劑
★ 肌肉
★ 代謝
★ 基因
關鍵字(英) ★ plasticizer
★ DEHP
★ MEHP
★ Skeletal
論文目次 Abstract I
中文摘要 II
I聲明 . III
目 錄 . IV
縮寫與全名對照表 縮寫與全名對照表 . VI

一 . 緒論 1
1-1 塑化劑 (Plasticizer) 1
1-2骨骼肌 (Skeletal muscle) 2
1-3 粒線體 粒線體 (Mitochondria) 2
1-4 TRPV1 (Transient Receptor Potential Cation Channel, Subfamily V, Member 1) 5

二. 實驗材料與方法 實驗材料與方法 . 7
2-1細胞培養 7
2-1-1細胞株 . 7
2-1-2 細胞培養 7
2-1-3穩定細胞株 7
2-1-4劑量之計算 7
2-2 動物試驗和 動物試驗和 飼養 . 7
2-2-1 實驗動物 8
2-2-2試驗物質 8
2-2-3 飼料配方置與投予法 配方置與投予法 8
2-2-4 保定法 . 8
2-2-4-1單手保定 單手保定 8
2-2-4-2 雙手保定 8
2-2-5 管餵法 . 9
2-2-6 動物犧牲、採血及組織保存 動物犧牲、採血及組織保存 9
2-3 Serum Analysis 10
2-3-1 Cholesterol Assay 10
2-3-2 Tg Assay 11
2-3-3 Glucose Assay 11
2-4 RNA Purification 12
2-4-1 Tissue RNA Purification 12
2-4-2 Cell RNA Purification 12
2-5 RNA Electrophoresis . 13
2-5 Reverse Transcription 13
2-6 genomic DNA Purification 14
2-7 PCR ( Polymerase Chain Reaction ) & DNA Electrophoresis 15
2-8 Q-PCR ( Quantitative Real Time Polymerase Chain Reaction ) 15
2-9 Cell Seeding On Coverslips 16
2-10 Fluorescence Microscope & Image J Quantitative 17

三. 結果.18
3-1 C2C12 在 2% HS 分化完整後的情況下,將細胞置換到含有 MEHP 的 20% FBS 下,肌管有萎縮趨勢 18
3-2 給予 MEHP 後大幅提升了 C2C12-CMB 粒線體 fission morphology 的現象 18
3-3 給予 MEHP 後不影響 C2C12 過氧化氫體的數量 . 19
3-4 給予 MEHP 後細胞代謝的路徑受到影響. 19
3-5 給予 DEHP 14 天後對於 wild type 和 TRPV-/- 的血液生化值沒有顯著影響 . 19
3-6 給予 DEHP 14 天後影響了和 TRPV1-/-的 mtDNA/ncDNA 的量 20
3-7 給予 DEHP 14 天後影響了 TRPV1-/- 其 gastrocnemius 對 PGC-1α 的調控以及其他代謝的traget gene 20
3-8、給予 DEHP 14 天後對於 wild type 、TRPV1-/-體重皆沒有太大差異 20

四 討論.22

五、圖表.28
圖一、C2C12 在 2%HS 分化完整後的情況下,將細胞置換到含有 MEHP 的 20% FBS 下,肌管
有萎縮趨勢 28
圖二、給予 MEHP 後大幅提升了 C2C12-CMB 粒線體 fission morphology 的現象 29
圖三、給予 MEHP 後不影響 C2C12 過氧化氫體的數量 30
圖四、給予 MEHP 後細胞代謝的路徑受到影響 32
圖五、給予 DEHP 14 天後對於 wild type 和 TRPV-/- 的血液生化值沒有顯著影響 33
圖六、給予 DEHP 14 天後影響了 TRPV1-/-的 mtDNA/ncDNA 的量 34
圖七、給予 DEHP 14 天後影響了 TRPV1-/- 其 gastrocnemius 對 PGC-1α 的調控以及其他代謝的traget gene 36
圖八、給予 DEHP 14 天後對於 wild type 、TRPV1-/-的體重皆沒有太大差異 38
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指導教授 陳盛良(Shen-Liang-Chen) 審核日期 2018-1-22
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