放線菌酮透過ERK蛋白激酶途徑刺激3T3-L1脂肪細胞內SOCS-3基因的表現

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蔡沛樺(Pei-hua Tsai) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

放線菌酮透過ERK蛋白激酶途徑刺激3T3-L1脂肪細胞內SOCS-3基因的表現
(Cycloheximide stimulated SOCS-3 gene expression in 3T3-L1 adipocyte via the ERK pathway)

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摘要(中)

SOCS-3 (suppressors of cytokine signaling-3) 蛋白質的分子量為24.7 kDa，在3T3-L1脂肪細胞上它會阻抗胰島素的訊息傳導。然而我們實驗室意外中發現：3T3-L1脂肪細胞在有cycloheximide (放線菌酮) 蛋白質合成抑制劑處理時， SOCS-3 mRNA的表現會上升，所以本論文目的是研究cycloheximide會如何影響脂肪細胞內SOCS-3 mRNA的表現。結果發現cycloheximide促進SOCS-3 mRNA的表現會因處理時間與劑量的不同而有差異，由於Actinomycin-D (RNA合成抑制劑) 會抑制cycloheximide的作用，顯示需要新的mRNA合成。有趣的是，在cycloheximide的處理中，細胞內SOCS-3的蛋白質表現並不會改變，其次，ERKs抑制劑例如U0126的處理，會減少cycloheximide增加的SOCS-3 mRNA表現量，顯示cycloheximide透過ERKs激酶相關的途徑去促進SOCS-3 mRNA表現。利用另一種蛋白質合成抑制劑例如anisomycin做處理，也會促進SOCS-3基因的表現。在不同的細胞株裡，cycloheximide對C3H10T1/2小鼠脂肪細胞和C2C12小鼠肌纖維母細胞也有促進SOCS-3 mRNA表現的作用，但不影響H4IIEC3大鼠肝腫瘤細胞內SOCS-3 mRNA的表現，顯示cycloheximide的作用有細胞特異性。我們另外發現cycloheximide會影響其它SOCS成員例如SOCS-1，SOCS-2，SOCS-4，SOCS-5，SOCS-6，SOCS-7及CIS 基因的表現，但高峰值出現的時間有所差異。最後，當處理5 μg/ml cycloheximide 6小時以內，它並不會造成前脂肪細胞和脂肪細胞的毒性，但當處理高劑量10 μg/ml 6小時以上或5 μg/ml 12小時以上，它會減少細胞數及存活率。綜合以上的結果，我們的結論：cycloheximide會透過ERKs激酶途徑調節脂肪細胞內SOCS-3 基因的表現。

摘要(英)

SOCS-3 (suppressors of cytokine signaling-3) is a 24.7 kDa protein that plays a very important role in the signaling transduction of insulin resistance caused by resistin in 3T3-L1 adipocytes. However, we found that cycloheximide, a protein synthesis inhibitor, could stimulate SOCS-3 mRNA expression in 3T3-L1 adipocytes. Despite of these, the exact mechanism of cycloheximide’s action on SOCS-3 gene expression and its effect on adipocyte are still unknown. Therefore, this study was to investigate how cycloheximide could affect the expression of adipocyte SOCS-3 gene. We found that cycloheximide stimulated SOCS-3 mRNA expression in a time- and dose-dependent manner. Actinomycin-D (a transcription inhibitor) blocked the cycloheximide-stimulated SOCS-3 mRNA expression, suggesting the need of a new mRNA synthesis, but not due to changes in the mRNA stability. Interestingly, cycloheximide did not significantly alter the SOCS-3 protein levels. Pretreatment with U0126 (an ERK MAPK inhibitor) reduced the cycloheximide-stimulated SOCS-3 mRNA levels by 50%. This suggests the ERK-dependent effect of cycloheximide. Treatment with an additional protein inhibitor, such as anisomycin, also stimulated SOCS-3 mRNA expression in C3H10T1/2 adipocyte and C2C12 myoblast, but it had no effect on H4IIEC3 hepatoma cell. This suggests that the cycloheximide effects vary with the cell types. Moreover, cycloheximide stimulated gene expression of other SOCS family members, such as SOCS-1，SOCS-2，SOCS-4，SOCS-5，SOCS-6，SOCS-7 and CIS；However, the different peak time of the individual SOCS mRNA level occurred among SOCSs. Finally, we found that the dose of cycloheximide at 10 μg/ml for 6-24 h and 5 μg/ml for 12-24 h, but not 5 μg/ml within 6 h, reduced cell number and cell viability of the preadipocytes and adipocytes. We conclude that cycloheximide stimulates 3T3-L1 adipocyte SOCS-3 mRNA expression via the MEK1/ERK-dependent pathway. Further study is needed to demonstrate whether the effects of cycloheximide on SOCS-3 gene expression are related to its action on fat cell viability.

關鍵字(中)

★ 3T3-L1脂肪細胞
★ SOCS-3基因
★ 放線菌酮

關鍵字(英)

★ 3T3-L1 adipocyte
★ SOCS-3
★ cycloheximide

論文目次

中文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥v
英文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥v i
誌謝‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ ‥vii
目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ ix
表目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ xi
圖目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ xii
縮寫與全名對照‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ xiii
第一章、緒論 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1
1. SOCS蛋白質‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 1
2. 放線菌酮 (Cycloheximide) ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 2
3. 研究動機與目的‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3
第二章、實驗材料與方法‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥5
2.1. 實驗材料‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥5
2.2. 細胞培養‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥5
2.3. 西方墨點法 (Western blotting assay)‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 7
2.4. 聚合酶鏈反應(PCR)‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
2.5. 細胞計算方法‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 10
2.6. 統計分析‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 10
第三章、結果‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
3.1. Cycloheximide在3T3-L1脂肪細胞中促進SOCS-3 mRNA表現‥‥‥‥12
3.2. Cycloheximide對SOCS-3蛋白質的影響‥‥‥‥‥‥‥‥‥‥‥‥‥ 13
3.3. Cycloheximide促進ERKs磷酸化，對JNK磷酸化沒顯著的影響‥‥‥‥13
3.4. Anisomycin促進3T3-L1脂肪細胞內SOCS-3 mRNA表現的現象‥‥‥15
3.5. Cycloheximide促進SOCS-3 mRNA的表現隨著不同細胞株而有差異性
的效果‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 15
3.6. Cycloheximide影響3T3-L1前脂肪細胞和脂肪細胞內不同型SOCS的 mRNA表現‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 16
3.7. Cycloheximide影響3T3-L1脂肪細胞內不同型SOCS的mRNA表現‥ 16
3.8. Cycloheximide對於3T3-L1前脂肪細胞和脂肪細胞存活率影響‥‥‥‥17
第四章、討論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18
4.1. 探討cycloheximide影響SOCS-3 mRNA‥‥‥‥‥‥‥ ‥‥‥‥‥‥18
4.2. 探討cycloheximide影響SOCS-3蛋白質的表現情形‥‥‥‥‥‥‥‥ 18
4.3. 探討cycloheximide是否透過ERK影響SOCS-3 mRNA表現‥‥‥‥‥19
4.4. Anisomycin促進3T3-L1脂肪細胞內SOCS-3 mRNA表現的現象‥‥‥20
4.5. 探討cycloheximide是否在其他細胞株也有相同效應‥‥‥‥‥‥‥‥ 20
4.6. 探討cycloheximide在3T3-L1前脂肪細胞和3T3-L1脂肪細胞影響其
它SOCS成員情形‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 21
4.7. 探討cycloheximide對細胞存活率的影響‥‥‥‥‥‥‥‥‥‥‥‥‥ 21
第五章、結論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
第六章、參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥24
第七章、圖表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥29
第八章、附錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥53

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指導教授

蔡沛樺、高永旭
(Pei-hua Tsai、Yung-Hsi Kao)

審核日期

2011-5-25

推文