辛基苯酚對3T3-L1脂肪細胞中resistin的調節作用

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吳敏華(Min-Hua Wu) 查詢紙本館藏

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生命科學系

論文名稱

辛基苯酚對3T3-L1脂肪細胞中resistin的調節作用
(Octylphenol regulation of resistin in 3T3-L1 adipocyte)

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摘要(中)

抗胰島素激素(resistin)是由脂肪細胞所分泌的荷爾蒙，其作用會降低了胰島素刺激脂肪細胞攝入葡萄糖的能力，而造成胰島素抗性，並且連結肥胖症和第二型糖尿病之間的關係。抗胰島素激素的表達量會受到許多荷爾蒙的調節，但整個調控機制仍然不清楚。然而環境荷爾蒙是由許多環境化合物所代謝產生，並且在累積中會影響到生物體內原本的生理活性，其中辛基苯酚(OP)結構和雌性素相似，並且會模仿雌性素的作用。故在本論文中，利用3T3-L1脂肪細胞做為研究細胞株，本研究發現OP會增加抗胰島素激素mRNA及蛋白質的產生，並且隨著OP的濃度及處理時間的不同而有程度上的差異。而在前處理轉錄抑制劑actinomycin D及轉譯抑制劑cycloheximide的情況下，OP刺激抗胰島素激素mRNA及蛋白質的表達量並不會受到改變，因此OP會增加抗胰島素激素mRNA及蛋白質的穩定性。實驗中也證實OP增加抗胰島素激素mRNA及蛋白質的表達會受到雌性素接受器抑制劑ICI182780及MEK1抑制劑U0126所抑制，顯示OP會經由ERα及MEK1訊息傳導途徑增加抗胰島素激素的產生，然而OP也會影響到ERα和抗胰島素激素負調節因子PPARγ之間的結合能力和PPARγ蛋白質表現，是否OP增加抗胰島素激素的產生之機制也與PPARγ的途徑有直接的關聯，將有待以釐清。

摘要(英)

Resistin (Rstn) is an adipocyte-specific secretory hormone that can reduce the insulin-stimulated glucose uptake by adipocytes, resulting in insulin resistance, and link the obesity and type II diabetes. It can be regulated by many hormones, but the exact mechanisms of their action is still not clear. Environment hormones are derived from the metabolites of many environmental chemical compounds and have the biological activity in organisms. Octylphenol (OP) is one of environmental estrogens that has the structural similarity to estrogen and can mimic the action of estrogens. In this study using 3T3-L1 adipocytes as the research cell line, OP induced increases in Rstn mRNA and protein expression with significantly dose- and time-dependent differences was discovered. Also, pretreatment with either transcriptional inhibitor actinomycin D or translational inhibitor cycloheximide did not prevent the OP-stimulated Rstn mRNA and protein expression. This suggests that OP increases the stabilities of Rstn mRNA and protein. In addition, pretreatment with either estrogen receptor alpha (ER?) inhibitor ICI182780 or MEK1 inhibitor U0126 prevented the OP-induced increases in Rstn mRNA and protein expression, suggesting that the effect of OP requires the functional ER? and MEK1. Moreover, OP increased PPAR? (a negative regulator of Rstn) protein expression and the association of ER? with PPAR?. Further studies to demonstrating whether the effect of OP on Rstn gene expression is mediated directly via modulation of PPAR? is needed.

關鍵字(中)

★ 辛基本酚
★ 抗胰島素激素
★ 脂肪細胞

關鍵字(英)

★ Octylphenol
★ resistin
★ adipocyte

論文目次

目錄 ------------------------------------------------ Ⅰ
圖目錄 ---------------------------------------------- Ⅳ
縮寫及全名對照表 ------------------------------------ Ⅴ
第一章:緒論 -------------------------------------------------------------- 1
一、抗胰島激素 (resistin) ------------------------------------------- 1
二、環境荷爾蒙(Environmental hormone) ------------------------------- 3
三、環境雌性素 ----------------------------------------------------------- 7
四、辛基苯酚 ------------------------------------------------------------- 8
五、雌性素影響resistin蛋白及基因的表達 ----------------------------------- 11
六、雌性素的分子作用機制與訊息傳導途徑 ----------------------------------- 12
七、研究動機問題與目的 --------------------------------------------------- 12
第二章 : 材料與方法 ------------------------------------------------------- 14
一、材料 ------------------------------------------------------------------ 14
二、實驗方法 -------------------------------------------------------------- 16
1. 細胞培養---------------------------------------------------------- 16
2. 細胞處理 --------------------------------------------------------- 16
3. 細胞計數方法 ----------------------------------------------------- 17
4. RNA 萃取---------------------------------------------------------- 17
5. 反轉錄酶反應(Reverse Transcriptase, RT) -------------------------- 18
6. 聚合酶鏈反應(Polymerase chain reaction, PCR) ----------------------18
7. PCR產物的分析 ---------------------------------------------------- 19
8. 蛋白質濃度定量 --------------------------------------------------- 20
9. 西方點墨法(Western blot analysis) -------------------------------- 20
10. 蛋白質免疫沉殿法 (Immunoprecipitation) --------------------------- 21
11. Enzyme-linked immunosorbent assay (ELISA) ------------------------ 22
12. 統計分析 --------------------------------------------------------- 23
第三章：結果 -------------------------------------------------------------- 24
一、 OP增加resistin mRNA表現及穩定度 ---------------------------------- 24
二、 OP增加resistin protein的表現 ------------------------------------- 24
三、 OP改變resistin表現的機制 ---------------------------------------- 25
1、 OP利用細胞原本的蛋白來產生resistin ------------------------------- 25
2、OP經由ERα的途徑改變resistin mRNA的表現 --------------------------------- 26
3、OP經由MAPK來促進resistin的表現 ----------------------------------------- 27
4、C/EBPα及PPARγ在OP促進resistin表現所扮演的角色--------------------------- 28
四、環境雌性素與雌性素對resistin的作用 ------------------------------- 29
五、 OP對3T3-L1細胞數目的影響 ----------------------------------------- 29
第四章：討論 -------------------------------------------------------------- 31
第五章：結論與展望 ------------------------------------------------------- 35
第六章：參考文獻 ---------------------------------------------------------- 36
第七章：附錄 -------------------------------------------------------------- 54
附錄一：溶液配制 ---------------------------------------- 54
附錄二：其他數據 --------------------------------------- 57
附錄三：Resistin DIG PCR ELISA ------------------------------------- 60

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指導教授

高永旭(Yung-Hsi Kao)

審核日期

2006-1-25

推文