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研究生:呂曉昀
研究生(外文):Hsiao-Yun Lu
論文名稱:抗胰島素蛋白對3T3-L1脂肪細胞脂解作用的影響與脂肪三酸甘油脂解酶及荷爾蒙敏感性脂解酶間關連性之探討
論文名稱(外文):The effects of resistin on 3T3-L1 adipocyte lipolysis and its involvement of adipose triglyceride lipase(ATGL) and hormone-sensitive lipase(HSL)
指導教授:何橈通何橈通引用關係
指導教授(外文):Low-Tone Ho, M.D.
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:63
中文關鍵詞:抗胰島素蛋白3T3-L1 脂肪細胞脂解作用脂肪三酸甘油脂解酶荷爾蒙敏感性脂解酶
外文關鍵詞:resistin3T3-L1 adipocytelipolysisATGLHSL
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脂肪分解(lipolysis)是一連串三酸甘油酯分解的過程。當此作用出現問題時,會破壞體內能量的平衡並造成胰島素阻抗。脂解作用受到許多荷爾蒙調控,如荷爾蒙敏感性脂解酶(Hormone-sensitive lipase, HSL),一種被認為在脂解作用中扮演速率限制步驟的酵素。在2004年,發現一個在脂肪組織中大量表現的新酵素,稱為脂肪三酸甘油脂解酶(adipose triglyceride lipase, ATGL),在油滴表面上會和其共同活化物CGI-58(又稱為Abhd5)結合,催化脂肪分解的起始步驟。抗胰島素蛋白(resistin)是一個在2001年發現的蛋白質,因其可拮抗胰島素的作用而命名之。根據研究顯示,resistin可以在體內及體外的狀況下引起脂肪細胞的脂解作用,但詳細的機轉目前並不瞭解。因此,本篇實驗目的在於探討ATGL以及HSL的表現,在resistin所引起的脂解作用中有何影響。實驗材料為3T3-L1前脂肪細胞分化成熟的脂肪細胞。細胞處理不同濃度的resistin(1、10、50、100ng/ml)及異丙腎上腺素(10-7M)不同時間(0、2、4、8、10及24小時)後分析培養液中甘油的濃度做為脂肪分解的參數,此外也分析ATGL及HSL基因與蛋白質的表現。結果顯示,以10ng/ml resistin處理3T3-L1脂肪細胞4小時可增加脂解作用,50ng/ml則會使ATGL及HSL表現量增加。resistin不會使HSL Ser660磷酸化。隨著10ng/ml resistin處理的時間增加,對細胞脂肪分解相較於control組沒有統計上的差異,而ATGL基因表現在4及10小時顯著下降,ATGL及HSL蛋白質的表現則沒有顯著影響;隨著10-7M異丙腎上腺素處理時間增加,ATGL基因表現會逐漸減少,對蛋白質表現則沒有顯著影響。HSL蛋白質表現量在10小時相較0小時表現量則顯著下降。cAMP的抑制物— 2’,5’-dideoxyadenosine(3X10-6M)可減少resistin及異丙腎上腺素所引起的脂解作用,且在有異丙腎上腺素的處理下會減少HSL蛋白質的表現,但對ATGL基因及蛋白質則沒有顯著影響。根據實驗結果顯示,resistin促進3T3-L1脂肪細胞的脂解作用可能不是透過調控ATGL及HSL的表現,且可能透過cAMP途徑刺激脂肪分解。因此,resistin刺激3T3-L1脂肪細胞的脂肪分解可能是透過影響ATGL的活性來達到刺激脂肪分解的目的,而非透過調控其蛋白質的表現量來影響。
Lipolysis is a process of triglycerides hydrolysis. Dysfunctional lipolysis disturbs energy homeostasis and contributes to obesity and insulin resistance. Lipolysis is regulated by numerous hormones and requires activation of lipolytic enzymes such as hormone-sensitive lipase (HSL), an enzyme presumed the rate-limiting enzyme for lipolysis. In 2004, a novel lipolytic enzyme, adipose triglyceride lipase (ATGL), was identified and found predominantly in adipose tissue. ATGL interacting with coactivator—CGI-58 (also called Abhd5) on the lipid droplet catalyzes the initial step of lipolysis. Resistin was identified in 2001 and named for resistance to insulin. Previous studies show that resistin can induce adipocytes lipolysis in vivo and in vitro, but the mechanism is still unclear. Thus, the present study was to characterize the expression of ATGL and HSL in resistin-induced lipolysis. 3T3-L1 preadipocytes were differentiated from fibroblasts to mature adipocytes and served as experimental materials. Cells were treated with different concentrations of resistin (1, 10, 50, 100ng/ml) and isoproterenol (10-7M) at various time periods(0, 2, 4, 8, 10, 24hr). Glycerol contents were measured as an index of lipolysis. Gene and protein expression of ATGL and HSL were examined. Resistin can stimulate 3T3-L1 adipocytes lipolysis at the 4hr incubation in 10ng/ml and increase ATGL and HSL protein expression compared with control in 50ng/ml, but has no effect on HSL Ser660 phosphorylation. 10ng/ml resistin has no effect on 3T3-L1 adipocytes lipolysis compared with control. It can decrease ATGL gene expression in 4 and 10hr treatment, but has no effect on both protein expressions. 10-7M Isoproterenol can decrease ATGL gene expression in 4, 8 and 24hr compared with 0hr, and decrease HSL protein expression in 10hr compared with 0hr. cAMP inhibitor—2’,5’-dideoxyadenosine (2’,5’-DD) can reduce resistin and Isoproterenol induced-lipolysis at the 4hr incubation but has no effect on ATGL gene and protein expression. Under 10-7M Isoproterenol treatment, 2’,5’-DD can reduce HSL protein expression. Our data suggested that the lipolytic effect of resistin might not through the regulation of ATGL and HSL expression. It might stimulate 3T3-L1 adipocytes lipolysis throught cAMP pathway. Thus, resistin stimulate 3T3-L1 adipocytes lipolysis may through activation of ATGL activity.
中文摘要 ………………………………………………………………………………1
英文摘要 ………………………………………………………………………………2
第一章、文獻回顧………………………………………………………………………3
一、脂肪分解…………………………………………………………………………3
(一)脂肪分解的調控…………………………………………………………3
(二)油滴表面的結構蛋白……………………………………………………5
(三)脂肪分解酵素……………………………………………………………6
二、抗胰島素蛋白 …………………………………………………………………11
三、研究目的與動機 ………………………………………………………………12
第二章、材料與方法…………………………………………………………………14
一、儀器設備………………………………………………………………………14
二、試劑藥品………………………………………………………………………14
三、實驗細胞………………………………………………………………………18
四、實驗方法………………………………………………………………………19
(一)脂肪細胞 Oil-Red O染色………………………………………………19
(二)3T3-L1脂肪細胞脂解分析………………………………………………19
(三)反轉錄聚合酶連鎖反應(reverse transcription-polymerase chain reaction, RT-PCR)……………………………………………………19
(四)西方墨點法(Western blot)……………………………………………21
五、統計分析 ………………………………………………………………………23
第三章、實驗結果……………………………………………………………………24
一、3T3-L1 脂肪細胞Oil-Red O 染色……………………………………………24
二、抗胰島素蛋白對3T3-L1脂肪細胞脂肪分解作用的影響……………………24
(一)劑量效應…………………………………………………………………24
(二)時間效應…………………………………………………………………24
三、抗胰島素蛋白對3T3-L1脂肪細胞中三酸甘油酯解酶(ATGL)及荷爾蒙敏感性脂解酶(HSL)基因與蛋白質表現的影響…………………………………25
(一)劑量效應…………………………………………………………………25
(二)時間效應…………………………………………………………………26
四、cAMP抑制物—2’,5’-dideoxyadenosine(2’,5’-DD)對抗胰島素白引起的3T3-L1脂肪細胞脂肪分解的影響 ……………………………………26
五、cAMP 抑制物2’,5’-DD 對3T3-L1脂肪細胞中三酸甘油酯解酶及荷爾蒙敏感性脂解酶基因與蛋白質表現的影響 ……………………………………27
第四章、討論 …………………………………………………………………………29
參考資料 …………………………………………………………………………… 34
附圖 ………………………………………………………………………………… 41
致謝 ………………………………………………………………………………… 60
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