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研究生:蔡宜蓉
研究生(外文):I-Jung Tsai
論文名稱:C5a於原發性兒童腎病症候群的角色:從臨床觀察到基礎研究
論文名稱(外文):The role of C5a in idiopathic nephrotic syndrome in children: from clinical to bench
指導教授:曹永魁曹永魁引用關係周祖述
指導教授(外文):Yong-Kwei TsauTzuu-Shuh Jou
口試委員:謝豐舟楊曜旭林石化陳念榮
口試委員(外文):Fong-Jou HsiehYao-Hsu YangShih-Hua LinNien-Jung Chen
口試日期:2015-05-05
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:臨床醫學研究所
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:2015
畢業學年度:103
語文別:英文
論文頁數:109
中文關鍵詞:兒童腎病症候群微小型變化C5aROCK pathway粘著連接 (adhesion junction)VE-cadherin肌動蛋白束(actin)張力絲(stress fiber)
外文關鍵詞:Nephrotic syndromeminimal change diseaseC5aROCKadherens junctionVE-cadherinactin stress fiber
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原發性腎病症候群 (idiopathic nephrotic syndrome) 為兒童常見的腎臟方面的疾病,主要的臨床表現為蛋白尿 (proteinuria)、白蛋白降低 (hypoalbuminemia)、高血脂 (hyperlipidemia)以及水腫 (edema)。根據International Study of Kidney Disease in Children的統計,罹患原發性腎病症候群的兒童中有76%在病理表現上為微小性變化(minimal change)。而約有93% 在病理變化上呈現微小性變化的腎病症候群的兒童,對於類固醇 (prednisolone)的標準療程具有很好的反應以及預後。針對原發性腎病症候群中的微小型變化,其致病的機轉至今仍未明。

在目前一系列有關腎臟的相關研究中,腎絲球足細胞 (glomerular podocyte) 在尿蛋白產生的可能機制占有重要的角色,傳統以來就被認定是蛋白尿的關鍵調控細胞。在一系列的研究設計與計畫中,我們探討了兒童原發性腎病症候群是否因為細胞性的免疫失調所造成的cytokine的變化程度不同,而導致對於治療上的反應有所不同。我們在臨床研究發現血清C5a在原發性腎病症候群的特殊表現,在病童發病時,血清中的有升高的趨勢。

我們進一步設計動物實驗,建立原發性腎病症候群的動物模式,將C5a注射至老鼠體內,引起老鼠出現蛋白尿,病理切片結果發現與微小性變化的腎病症候群相近。進一步再藉由純化腎絲球內皮細胞 (glomerular endothelial cells),利用體外細胞模式,確認C5a受器 (C5a receptor, C5aR)主要表現於腎臟血管內皮細胞,而非位於足細胞。再進一步探討C5a會如何影響腎絲球內皮細胞 (glomerular endothelial cells),我們發現C5a藉由Rho/Rho-associated kinase/myosin途徑的活化,造成內皮細胞的收縮 (endothelial cell contraction),造成細胞間隙變大 (cell adhesion complex breakdown),引起蛋白尿的表現。此外,進一步在細胞實驗加入Rho kinase pathway抑制劑 (Rho-associated kinase inhibitor, Y27632),結果發現在施打C5a的老鼠引起的蛋白尿,其蛋白尿有減少情形。

因此,在一系列從臨床的觀察,發現原發性腎病症候群的病童,其血清C5a有升高的趨勢。建立動物模式,了解C5a可能為引起蛋白尿的因子,進一步利用細胞實驗,確定C5a是透過Rho/Rho-associated kinase/myosin pathway影響腎絲球內皮細胞通透能力,為造成蛋白尿的可能機制。我們的研究提供了對於原發性腎病症候群的致病機轉新的解釋,重新定義腎臟內皮細胞於蛋白尿形成過程中可能扮演的角色,並提供C5a影響內皮細胞通透性的新的訊息傳遞機轉。進一步,也期待藉由此分子訊息傳導的機轉的發現,能夠作為未來在治療原發性腎病症候群的新的治療選擇。

Childhood idiopathic nephrotic syndrome (INS) is mainly caused by minimal change disease because only subtle ultrastructural alteration could be observed at electron microscopic level in the pathological examination. Glomerular podocytes are currently presumed to be the target cells in nephrotic syndrome whose protein sieving capability is compromised by a yet unidentified permeability perturbing factor.

For the majority of INS cases in children, they manifest minimal change disease and lack apparent direct targets specific for podocytes. Other components of the glomerular protein sieving apparatus, such as the glomerular endothelial cells might play any functional role in childhood nephrotic syndrome remains to be undetermined.

INS pediatric patients who were diagnosed in National Taiwan University Hospital during 2008-2012 were enrolled in this study and a human cytokine antibody array was exploited to analyze the cytokine profiles. In animal model establishment, six-week-old male ICR mice were treated with normal saline and recombinant mouse C5a respectively with the dose of 10 μg/kg every three days through tail vein injection. Blood and urine samples were collected. After 21 days, mice were sacrificed for pathology study. The glomeruli were isolated from minced kidneys, and the kidney endothelial cells bound to the magnetic beads were isolated and preparing for protein permeability assay on endothelial monolayer, immunofluorescence and F-actin staining and GTP-Rho pull-down assay. All the data were expressed as mean ± SD. Comparisons for continuous data between relapse and remission in INS patients were made by paired t-test. In animal studies, a two-tailed t-test was used in analysis of biochemical data between two groups. In cell study, a two-tailed t-test was statistically significant differences between two groups. A p < 0.05 was statistically significant cut-off value.

In a cohort study of INS children, we found the complement fragment C5a was elevated in their sera during relapse status. Administration of recombinant C5a induced profound proteinuria and minimal change nephrotic syndrome in mice. Purified glomerular endothelial cells, instead of podocytes, were demonstrated to be responsible for the proteinuric effect elicited by C5a. Further studies depicted a signaling pathway involving Rho/Rho-associated kinase/myosin activation leading to endothelial cell contraction and cell adhesion complex breakdown. Significantly, application of Rho-associated kinase inhibitor, Y27632, prevented the protein leaking effects observed in both C5a-treated purified endothelial cells and mice.

Our study identifies a previously unknown mechanism causing pediatric INS and provides a new insight toward identifying Rho-associated kinase in kidney endothelial cells as a possible pathogenesis induced proteinuria in INS and a considerable alternative therapeutic option for children with nephrotic syndrome.


口試委員會審定書………………………………………………….……………
誌謝………………………………………………………………………….…… i-ii
中文摘要……………..…………..…………………………………….………… iii-iv
英文摘要………………...………………………………………..……………… v-vi
博士論文內容
1. 緒論 (Introduction) ……………………………………………………….… 1-22
2. 研究方法與材料 (Material and Methods) ………………………………….. 23-29
3. 結果 (Results) ………………………………………………………………. 30-36
4. 討論 (Discussions) ………………………………………………………….. 37-52
5. 展望 (Expectation) ……………..…………………………………………… 53-63
6. 參考文獻 (References) ……………………………………………………... 64-80
表1~表4…..…………………………………………………………………….. 82-85
圖1~圖20…….…………………………………….……………………………. 86-108

附錄:列出個人在碩博士班修業期間所發表之相關論文清冊……………… 109


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