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Title: | 以蛋白質體學探討糖尿病相關腎臟病變及胰臟胰島異常之分子機制 Studies on Molecular Mechanisms of Diabetes-related Nephropathy and Pancreatic Islet Abnormality Using Technology of Proteomics |
Authors: | Siao-Syun Guan 官孝勳 |
Advisor: | 劉興華 |
Keyword: | 糖化終產物,糖尿病腎病變,腎纖維化,ATP5b,β細胞,細胞肥大,熱休克蛋白60, advanced glycation end products,diabetic nephropathy,ATP5b,renal fibrosis,β-cell hypertrophy,heat shock protein 60, |
Publication Year : | 2016 |
Degree: | 博士 |
Abstract: | 腎臟病變與胰島β細胞功能異常是高血糖狀態下常見的併發症。高血糖會促使糖化終產物 (advanced glycation end products, AGEs) 生成,並堆積在糖尿病患者大部分的組織。 AGEs引發ROS的產生,並活化訊息傳遞路徑去誘導腎臟纖維化與胰島β細胞肥大,但對於AGEs促使併發症生成的分子機制仍不清楚。因此,本篇論文是利用蛋白質體學的技術搜尋出與兩個與AGEs引發併發症相關的蛋白質-ATP合成酶β次單元 (ATP synthase subunit-β, ATP5b) 與熱休克蛋白60 (heat shock protein 60, HSP60),並分成兩個部分進行探討。
第一部分,我們將研究ATP5b在糖尿病腎病變所扮演的角色。與對照組相比,我們觀察到糖尿病的小鼠,其組織型態產生改變、纖維化、α-平滑肌肌动蛋白 ( α-smooth muscle actin, α-SMA)、AGEs和ATP5b有明顯的增加。ATP5b主要表現在腎小管的位置,但腎絲球的部位不明顯。AGEs透過腎小管細胞表面對應的接受器 (the receptor for AGEs, RAGE),促使ATP5b及纖維化蛋白的明顯的表現,並降低細胞ATP的含量。而在糖尿病小鼠的腎臟組織及AGEs處理的後的腎小管細胞皆有觀察到氧化壓力明顯增加。然而,抗氧化藥物N-acetylcysteine可以抑制AGEs誘導ATP5b以及結締組織生長因子 (connective tissue growth factor, CTGF) 蛋白質表現增加的現象。此外,當腎小管細胞的ATP5b表現被抑制後,在AGEs的作用下,會使α-SMA and CTGF表現更加顯著,且 CTGF promoter 活性也增加。最後將糖尿病小鼠的腎臟ATP5b表現抑制後可發現,血清肌酸酐以及腎臟纖維化的現象更加明顯。上述結果顯示,在糖尿病的狀態下,大量表現ATP5b對於 AGEs造成的腎臟病變具有保護的角色。 第二部分,我們要探討HSP60在糖尿病引起的胰島β細胞肥大及功能缺陷所扮演的角色。HSP60是一種粒線體的伴護子,而AGEs已經被認為會對β細胞功能造成影響。我們假設第二型糖尿病引起的β細胞肥大現象是透過HSP60之異常調控路徑,使得AGEs誘導細胞肥大及功能缺失。我們利用糖尿病小鼠以及β細胞去研究 AGEs誘導細胞肥大與功能異常中HSP60所扮演的角色。與對照組相比,糖尿病患者的胰臟切片顯示,胰島部位有肥大、AGEs含量增加以及HSP60表現下降的現象。而在12週大的糖尿病小鼠也發現細胞肥大標記p27Kip1增加、AGEs和RAGE表現增加,而HSP60、胰島素與ATP含量皆下降。當β細胞給予RAGE抗體中和反應後,低濃度AGEs所造成的細胞肥大、p27Kip1表現增加、HSP60表現下降、胰島素分泌與ATP含量降低的現象皆被抑制。大量的HSP60表現可呈現細胞保護的效果,進而去抵抗AGEs所誘導的β細胞肥大、功能異常以及ATP含量下降。另外,AGEs降低β細胞的HSP60表現也與氧化壓力有關。綜合上述,這些發現顯示,當在糖尿病的態下,HSP60可能是一個做為AGE-RAGE訊息傳遞誘導β細胞肥大與功能異常的標的物。 Diabetic nephropathy and pancreatic β-cell dysfunction are the major complications caused by hyperglycemic condition. Hyperglycemia increases formation of advanced glycation end products (AGEs) and accumulates to most of tissue in diabetes patients. AGEs induce ROS generation and activation of signal transduction to cause renal fibrosis and islet β cell hypertrophy, but the mechanisms are still unclear. Here we investigated the relation of factors and mechanism which was involved in AGEs caused renal fibrosis and β-cell hypertrophy and dysfunction in type 2 diabetes (T2D) mice. Moreover, we utilized proteomics technique to find out two proteins –ATP synthase subunit-β (ATP5b) and heat shock protein 60 (HSP60) which were involved AGEs-induced diabetic nephropathy and pancreatic β-cell dysfunction, respectively. Here, we divided two parts of this study for discussion. In the first part of this study, we investigated the role of ATP5b in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), AGEs, and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via a AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and connective tissue growth factor (CTGF) protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. Moreover, ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. Finally, the in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs induced renal fibrosis during diabetic condition. In the second part of this study, we investigated the role of HSP60 in pancreatic islet hypertrophy and dysfunction during hyperglycemia condition. HSP60 is a mitochondrial chaperone. AGEs have been shown to modulate the β-cell function. We hypothesized that AGEs induced β-cell hypertrophy and dysfunction through a HSP60 dysregulation pathway during the stage of islet/β-cell hypertrophy of T2D. We investigated the role of HSP60 in AGEs-induced β-cell hypertrophy and dysfunction using the models of diabetic mice and cultured β-cells. Pancreatic sections from diabetic patient showed islet hypertrophy, increased AGEs level, and decreased HSP60 level as compared with normal subject. Hypertrophy, increased levels of Cyclin-dependent kinase inhibitor 1B (p27Kip1), AGEs, and RAGE, and decreased levels of HSP60, insulin, and ATP content were obviously observed in pancreatic islets of 12-week-old db/db diabetic mice. Low-concentration AGEs significantly induced the cell hypertrophy, increased the p27Kip1 expression, and decreased the HSP60 expression, insulin secretion, and ATP content in cultured β-cells, which could be reversed by RAGE neutralizing antibody. HSP60 overexpression showed cytoprotective effects against AGEs-induced hypertrophy, dysfunction, and ATP reduction in β-cells. Moreover, oxidative stress was involved in the AGEs-decreased HSP60 expression in β-cells. Taken together, these findings highlight a novel mechanism by which HSP60 is a possible target for AGEs-RAGE axis-induced β-cell hypertrophy and dysfunction under diabetic hyperglycemia. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51282 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 毒理學研究所 |
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