2014-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647847摘要：原發性腎病症候群(idiopathic nephrotic syndrome)為小孩子常見的腎臟疾病，我們先前以經由臨床檢體發現 cytokine C5a可能與此病症有關，也進一步發現會 C5a確實會造成小鼠蛋白尿。本研究計畫主要延續前一年度的計畫；在去年度的計畫中我們運用 C5a誘發動物蛋白尿模式，配合蛋白質體學分析再以生物資訊學的研究方式，以系統生物學的模式探討 C5a誘發動物蛋白尿模式的分子機制。目前已經得到的實驗結果為 1. 運用蛋白質體學分析在 C5a 誘發之蛋白尿動物模式中的腎臟組織之蛋白質表現情況。2. 運用 GC-MASS 鑑定 300個在控制組與實驗組中具差異性的蛋白質身份。3. 運用具差異性蛋白質的生物資訊學資料進行系統生物學分析。 在本二年期計畫中我們主要將延續此蛋白質-系統生物學的研究結果，以細胞生物學與實驗動物探討下列問題； 第一年主要的目標是以細胞生物學確認 C5a對目標蛋白質的作用機制。我們已經確認 C5aR 主要表現於腎臟血管內皮細胞。因此細胞學的研究，將以初代培養的小鼠腎臟 endothelial cells 來進行；研究策略與方法為: 1. 由蛋白質身份鑑定結果，我們篩選出與 cell junction 功能有關的 10個細胞骨架分子；將進一步以免疫螢光染色確認特殊蛋白質在腎臟組織中，尤其是 endothelial cells 上的表現位置。 2. 以外加 C5a重組蛋白的方式，確認特殊蛋白質在表現量與座落位置上受影響的情況。 3. 將以 siRNA抑制特殊細胞骨架分子，驗證其在細胞通透能力的角色。 4. 以 Receptor tyrosine kinase protein array探討 C5a作用之特殊細胞骨架分子所活化之細胞訊息傳遞。 第二年主要的目標是以小鼠模式確認 C5a調控之細胞通透能力的分子機制，並探討以 C5a調控之相關細胞訊息傳遞分子機制，作為治療 C5a誘發小鼠蛋白尿的分子標靶治療的可行性。研究策略與方法為: 1. 以組織免疫螢光染色，確認在不同時間點，C5a誘發小鼠蛋白尿模式中，特殊細胞骨架蛋白質在腎臟組織中的表現量與座落位置的情況。 2. 以組織免疫螢光染色，確認在不同時間點，C5a誘發小鼠蛋白尿模式中，特殊訊息傳遞分子之磷酸化在腎臟組織中的表現情況。 3. 以 C5a誘發小鼠蛋白尿模式，探討特殊訊息傳遞抑制劑對此模式蛋白尿模式的預防及治療效果。<br> Abstract: Nephrotic syndrome is a common renal disease in children and it is defined as proteinuria, hypolbuminemia, hyperlipidemia and edema. Our previous studies have demonstrated the relation between idiopathic nephrotic syndrome and serum level C5a. Furthermore, we also find that tail vein injection of recombinant mouse C5a did cause proteinuria in mouse. In this project, we will continue the previous project to study the molecular mechanism of C5a mediated proteinuria. In previous project, we used the proteomics and system biology to investigate the C5a targeting protein in mouse kidney. We have identified 300 proteins with differential expression, based on the bioinformation and system biology analysis. We found that C5a may affect cytoskeleton protein of kidney. In this two years project, we will confirm the expression and the roles of 10 identified proteins that are screen out by the previous study by molecular cell biology and animal study. The aim of the first year is to confirm the mechanism of C5a on targeting protein by cell biology. Our preliminary data has revealed that C5aR is expressed in isolated endothelial cells from kidney but not in podocytes. Based on this finding, primary culture of kidney endothelial cells will be the cell model for the following studies. The study designs and major methodologies are: 1. Based on the protein identification data, 10 proteins that with function of cell junction and cytoskeleton will be firstly verified its expression and location in kidney especially on endothelial cells by Immunofluorescence staining. 2. To confirm the expression and location of specific protein by treating primary endothelial cells with recombinant C5a. 3. To inhibit the specific protein expression by siRNA strategy and following evaluate its role on the cell permeability. 4. To evaluate the specific signaling transduction that mediated by C5a activated cytoskeleton by receptor tyrosine kinase protein array. The aim of the second year is to confirm the mechanism of C5a mediated endothelial permeability in vivo. Furthermore, we will clarify the therapeutic potential of C5a related signaling transduction inhibitors on the C5a induced proteinuria mouse model. The study designs and major methodologies are: 1. To verify the time course effect of C5a mediated dynamic change of specific cytoskeleton in the kidney from C5a induced proteinuria mice by Immunofluorescence staining. 2. To verify the time course effect of C5a mediated phosphorylation change of specific signaling transduction mediators in the kidney from C5a induced proteinuria mice by Immunofluorescence staining. 3. To clarify the therapeutic effect and prevention effect of C5a related signaling transduction inhibitors on the C5a induced proteinuria mouse model.non-small cell lung cancer (NSCLC)a disintegrin and metalloprotease 15 (ADAM15)Src homology (SH)3 domain