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研究生:王作萍
研究生(外文):Tso-Ping Wang
論文名稱:脂肪細胞分化調控與基因傳送之研究
論文名稱(外文):The differentiation and gene delivery of adipocytes
指導教授:戴明泓
指導教授(外文):Ming-Hong Tai
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:89
中文關鍵詞:脂肪酸合成酶脂肪生成作用分化作用肥胖症複製擴增
外文關鍵詞:mitotic clonal expansionobesityfatty acid synthetasedifferentiationadipogenesis
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  • 被引用被引用:2
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最近的調查報告指出,近年來肥胖問題日益嚴重,台灣目前大約就有四百萬人的體重過重。WHO及美國疾病控制與預防中心曾公告:肥胖將是本世紀的最大健康殺手,它對健康的傷害程度可與香菸相比。肥胖不但會帶來心臟病、糖尿病、心血管疾病與高血壓、並會提高癌的罹患率、加速罹患阿滋海默症、膽囊疾病與縮短壽命等。然而由於導致肥胖的脂肪代謝缺陷機轉極為複雜,因此針對脂肪細胞分化方面的分子調控機制研究亦便愈來愈被重視。
脂肪生成作用(adipogenesis),指的便是經由分化形成脂肪組織之過程,此生化途徑經由許多實驗、應用許多脂肪細胞株研究,目前已較被了解。而來自老鼠的3T3-L1細胞株,是其中最具代表性且應用最廣泛的細胞株。已知脂肪細胞分化的過程是由一系列事件所驅動,其前身細胞初似長梭狀成纖維母細胞(fibroblast),並無脂質呈現,經由誘發物質Dexamethasone、Isobutylmethylxanthine(IBMX)和遠超出生理濃度之Insulin等協同作用,五天後便會轉形,變成堆積大量脂質且體積變大變圓的脂肪細胞。其中Dexamethasone會活化轉錄因子CCAAT/enhancer-binding protein β(C/EBPβ),IBMX會抑制可溶性 cyclic nucleotide phosphodiesterases,結果使細胞中cAMP 量增加。同時IBMX也會活化細胞核中相關轉錄因子C/EBPδ, C/EBPβ和C/EBPδ會使C/EBPα和PPARγ開始轉錄。誘發後三天內,脂肪細胞開始進行兩次有絲分裂,亦即脂肪細胞分化所需的複製擴增(mitotic clonal expansion)過程。Insulin或insulin-like growth factor-1 經由活化PI 3-kinase和Akt作用途徑,以及調節forkhead 轉錄因子 Foxo1活性,亦能加速脂肪細胞分化。C/EBPα和PPARγ經由活化脂肪細胞特異基因表現,例如脂肪酸合成酶(fatty acid synthetase)、脂肪酸結合蛋白(fatty acid binding protein)、leptin 和adiponectin,而直接作用於脂肪生成作用的最後階段。因此期望能對脂肪生成作用中之各調節產物有更深一層的了解,進而可利用相關藥物徹底避免並治療肥胖症。本論文之研究重點在探討老鼠3T3L-1前脂肪細胞與人類PLA脂肪細胞(processed lipoaspirate cells)於細胞分化與增生的調節機制,利用所建立的體外培養脂肪細胞方法,配合誘發脂肪生成與基因傳送技術,進一步研究肥胖相關基因(POMC與PTEN)及下游調節產物(PPARγ、adiponectin等)在脂肪細胞分化過程中所扮演的角色。
As shown by recent reports, number of obese people in recent years has been on the increase, there are about 4 million people in Taiwan who are considered to be overweight. World Health Organization (WHO) and United States Center for Disease Control and Prevention (CDC) publicly announced that: Obesity will be the greatest health killer of this century, its damage to personal health is comparable to that of cigarettes. Obesity can cause heart problems, diabetes, artery diseases, high blood pressure, increased chances of cancer occurrence, condition increase and deteriora- tion of Alzheimer’s disease, gall bladder diseases, and shortening of life span. The cause of obesity is due to a fault in adipocytes metabolism functions, and because of this, research into adipocytes molecular regulation is becoming more popular and valued. The process of adipogenesis, the formation of adipose tissue, has become better understood by the studies of several cell types that can be induced to undergo differentiation into adipocytes. The first, and the best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer -binding protein β (C/EBPβ). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBPδ. Immediately after exposure to exogenous inducers, the gene expression of C/EBPβ and C/EBPδ significantly and transiently increases, C/EBPβ and C/EBPδ may also regulate the expression of C/EBPα and PPARγ. C/EBPα and PPARγ are considered to play a prominent role in regulating the gene expression of proteins necessary for the development fo the functional mature adipocyte. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBPα and PPARγ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin. The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. My research is aimed at investigating the adipocytes differentiation and regeneration adaptive mechanisms of mice 3T3L-1 preadipocytes and human processed lipoaspirate cells (PLA). By using adipocytes culture techniques in conjunction with adipocytes growth induction and gene delivery techniques to further study obesity related genes, POMC and PTEN, and downstream regulators , PPARγ and Adiponectin, in regards to their roles in the process of adipocytes differentiation.
目錄 頁次
摘要 1
關鍵詞 2
英文摘要 3
緒言 5
背景及重要性 5
脂肪細胞 5
前脂肪細胞分化過程中Mitotic Clonal Expansion的機制 7
3T3-L1脂肪前驅細胞 7
脂肪細胞的分化誘導 8
幹細胞(stem cells) 9 全能性幹細胞(totipotent stem cells) 9
多能力性幹細胞(pluripotent stem cells) 10
多潛能幹細胞(multipotent stem cells) 11
單能幹細胞(unipotent stem cells) 12
脂肪組織來源幹細胞(processed lipoaspirate cells,PLA) 13
Wortmannin經PI3K途徑抑制 脂肪合成 14
PPARγ 與脂肪細胞分化 15

頁次
脂肪細胞分泌產物Adiponectin 16
腺病毒基因傳送研究 17
建構重組腺病毒載體 18
前-腦啡-黑細胞促素-皮促素(POMC) 22
腫瘤抑制基因PTEN 24
實驗設計 26
研究方法 28
1. 細胞株之培養 28
(1) 293 細胞株之培養 28
(2) 3T3-L1脂肪前驅細胞株(Swiss 3T3 mouse cell)之培養 28
(3) PLA(processed lipoaspirate cells)細胞株之培養 29
2. 細胞株之分化 29
(1) 3T3-L1脂肪前驅細胞株之分化 29
(2) PLA細胞株之分化 30
3. 細胞活性之測定 30
(1) MTT細胞增殖試驗(Cell Proliferation Assay) 30
(2) 錐蟲藍(trypan blue)染色法 31
4. Oil Red O染色 32
頁次
5. 測量波長的選擇 32
6. 蛋白質濃度分析 33
7. 蛋白質電泳分析 33
8. 西方墨點法 34
9. 脂肪細胞核酸之萃取 35
10. 反轉錄作用合成cDNA 36
11. 即時定量聚合酶鏈鎖反應(Real-time PCR) 36
12. 分離RNA並以RT-PCR選殖人類POMC之cDNA 38
13. 建構重組腺病毒 38
14. 量產及純化重組腺病毒 39
15. 病毒效價分析(Quick CPE Assay) 40
16. 測定腺病毒感染脂肪細胞之最適感染條件 41
17. 以重組腺病毒感染脂肪細胞 42
18. 偵測脂肪細胞分泌產物Adiponectin 42
19. ACTH 分析 42




頁次
結果 44
1. 建立老鼠與人類脂肪細胞株之培養與分化條件 44
2. 評估以吸光值觀察脂肪細胞分化之可行性 44
3. 評估腺病毒在老鼠與人類脂肪細胞之基因傳送效率
與最佳感染條件 46
4. 觀察重組腺病毒基因傳送影響脂肪細胞生長分化情形 47
5. 觀察脂肪細胞經重組腺病毒感染後,釋放adiponectin
之表現量比較 48
討論 50
參考文獻 55

圖目錄 頁次
圖一:3T3-L1脂肪細胞分化前後,以Oil Red O染色分析 58
圖二:3T3-L1前脂肪細胞之代謝與分化訊息傳遞途徑 59
圖三:利用分光光度計測定Oil Red O染液最大吸光度 60
圖四:Wortmannin對3T3-L1脂肪細胞於分化前後之影響 61
圖五:Wortmannin對3T3-L1脂肪細胞於分化前後之影響 62
圖六:重組腺病毒基因傳送流程圖 64
圖七:重組腺病毒之製作及POMC重組腺病毒確認 65
圖八:測試PLA細胞之最適腺病毒感染條件 67
圖九:前-腦啡-黑細胞促素-皮促素(POMC) 68
圖十:PLA細胞(processed lipoaspirate cells)分化結果 69
圖十一:POMC基因重組腺病毒感染PLA細胞 70
圖十二:PTEN蛋白結構及抑制PI 3-Kinase訊息傳遞途徑 72
圖十三:PTEN基因重組腺病毒感染PLA細胞 74
圖十四:PTEN基因重組腺病毒感染PLA細胞 76
圖十五:基因重組腺病毒對脂肪細胞分化之影響 77
圖十六:基因重組腺病毒對脂肪細胞分化之影響 79
圖十七:基因重組腺病毒對脂肪細胞分化之影響 81


頁次
圖十八:以Real-Time PCR測試脂肪細胞經重組腺病毒
感染後,釋放adiponectin之表現量比較 83
圖十九:ELISA法測試adiponectin之表現量比較 84
圖二十:POMC基因重組腺病毒轉染脂肪細胞後,
以西方墨點法測試PPARγ之表現 85


























表目錄 頁次
表一:PLA細胞經過不同組成培養液誘導分化處理後,
生長成不同類型之新人體細胞 87
表二:分別配製如表列濃度之Oil Red O染料,選擇最大
吸收波長 88
表三:即時定量聚合酶連鎖反應(Real-time PCR),測試
脂肪細胞經基因重組腺病毒感染後,adiponectin的
基因轉錄表現值 89
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