- 學位論文
大白鼠腎上腺皮質細胞內貝它型肌動蛋白與細胞內脂肪滴連結之功能研究
Functional Studies of the Association Between beta-actin and Lipid Droplets in Rat Adrenocortical Cells
摘要
中文摘要 細胞內脂肪滴表面的蛋白質，多數具有調節脂質代謝之功能。然而細胞內脂質代謝之機轉及運送途徑仍有許多未知，所以本實驗擬探討是否仍有其他的脂肪滴表面蛋白質存在，以及其功能為何。本實驗取材自大白鼠腎上腺皮質細胞或脂肪細胞，分離出細胞內之脂肪滴，利用電泳(SDS-PAGE)與免疫轉漬方法分析脂肪滴表面的蛋白質，結果發現，不論是從腎上腺皮質細胞或脂肪細胞分離出的脂肪滴中，皆有肌動蛋白(actin)存在；經二次電泳及免疫轉漬方法，證實為貝它型肌動蛋白(b-actin)。進一步進行腎上腺皮質細胞培養，利用免疫螢光染色觀察細胞內b-actin分佈的位置，結果顯示b-actin除了架構成肌動蛋白絲(actin filaments)之外，同時也分佈於脂肪滴表面。另以FITC-phalloidin的染色方法證實脂肪滴表面的b-actin為顆粒狀(globular type)。此外，再分別利用藥物dibutyryl cyclic AMP促使細胞內的脂質水解；或藥物cytochalasin D破壞細胞內的肌動蛋白絲(actin filaments)，而後再藉由免疫螢光染色觀察脂肪滴的形態，以及其表面的肌動蛋白分佈位置的變化。實驗結果發現，以dibutyryl cyclic AMP刺激，會加速細胞脂質分解及固醇類物質的分泌，且脂肪滴會隨著藥物刺激時間的增加而變小，而原本位於脂肪滴表面的b-actin會脫落並游離於細胞質中。而加了藥物cytochalasin D後細胞明顯地皺縮及肌動蛋白絲斷裂瓦解，β-actin則明顯堆積於脂肪滴的表面，似乎有保護脂肪滴的功能，使之不被甲醇溶解。因此推測 b-actin附著於脂肪滴表面，可能具有保護脂肪滴避免被破壞或被水解之功能。
並列摘要
Abstract Several lipid droplet-associated proteins are involved in regulating the intracellular lipid metabolism. However, the molecular basis of the acute hormonal regulation and mechanism of lipolysis in cells remains unclear. In this study, intracellular lipid droplets were isolated from rat adrenocortical cells and adipocytes. The lipid droplet-associated proteins were analyzed by electrophoresis and Western blotting. We found that actin(43kD) was copurified with both lipid droplet preparations. According to the data of one and two dimensional electrophoresis and Western blot, lipid droplet-associated actin was confirmed as beta isoform. Immunofluorescence with anti-b-actin antibody showed that b-actin not only constructed the stress fibers, but also located on the surface of lipid droplet. In addition, FITC-phalloidin staining displayed lipid droplet-associated actin was globular type. To investigate the interaction between b-actin and intracellular lipid droplets, the redistribution of b-actin was examined by administration of dibutyryl cyclic AMP and cytochalasin D. After treatment of dibutyryl cyclic AMP to stimulate lipolysis and steroidogenesis, lipid droplets were decreased in size. Immunofluorescence with anti-b-actin antibody revealed that the number of stress fibers were decreased and b-actin was translocated from the surface of lipid droplets into cytoplasm in cultured adrenocortical cells. After treatment of cytochalasin D to depolymerize actin filaments, scattered b-actin granules were observed in cytosol and each methanol-resistant lipid droplet was surrounded by a bright rim. These data demonstrated that the lipid droplet-associated globular b-actin might protect lipid droplets from methanol extraction or lipolysis.
參考文獻
延伸閱讀
- 賴豐傑（2002）。Deoxycorticosterone Acetate-salt 處理後大白鼠腎上腺皮質及髓質ANP合成能力增加。中華民國內分泌暨糖尿病學會會訊，15(2)，29-32。https://www.airitilibrary.com/Article/Detail?DocID=a0000018-200201-15-2-29-32-a
- 蔡佩芸（2010）。大鼠腎臟中糖尿病相關蛋白之蛋白質體之螢光高效能液相層析串聯質譜分析〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://doi.org/10.6831/TMU.2010.00066
- Ho, W. H., Kao, M. H., Huang, B. N., Chang, H. C., & Lu, K. S. (1994). 倉鼠腎上腺髓質之含顆粒小（SGC）細胞. The Kaohsiung Journal of Medical Sciences, 10(1), 28-34. https://doi.org/10.6452/KJMS.199401.0028
- Lin, M. H. (2014). Functional and Structural Studies of Lipoprotein LipL41 from Pathogenic Leptospira [doctoral dissertation, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-2912201413490807
- Martin, S., Veen, S. V., Holemans, T., Demirsoy, S., Haute, C. V. D., Baekelandt, V., Agostinis, P., Eggermont, J., & Vangheluwe, P. (2016). Protection against Mitochondrial and Metal Toxicity Depends on Functional Lipid Binding Sites in ATP13A2. Parkinson＇s Disease, 2016(), 689-699. https://doi.org/10.1155/2016/9531917