Analysis of molecular mechanism and global gene expression on urolithiasis using cDNA array

利用cDNA芯片分析尿石症的分子机制和整体基因表达

• 批准号: 13671678
• 负责人: MIYAZAWA Katsuhito
• 金额: $ 2.24万
• 依托单位: KANAZAWA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671678/
• 关键词: urolithiasis; Calcium oxalate; CDNA array; Gene expression; Renal epithelial cell; Potassium oxalate; DNAマクロアレイ

Purpose :Kidney stone formation is a complex process, and numerous genes participate in this cascade. The binding and internalization of calcium oxalate monohydrate (CON) crystals, the most common crystal in renal stones by renal epithelial cells may be a critical step leading to kidney stone formation. Exposure to CON crystals alters the expression of various genes, but previous studies on gene expression have generally been limited. To obtain more detailed insight into gene expression, we examined gene expression profiles in renal epithelial cells exposed to CON crystals using cDNA macroarray. Materials and Methods : NRK-52E cells were exposed to CON crystals for 60 and 120 minutes. Poly (A)^+ RNA was isolated and converted into ^<32>P-labeled first-strand cDNA, then the cDNA probe was hybridized to the membrane. Hybridization images were scanned and the signal intensities were quantified. Expression of mRNA of 1176 genes were analyzed with global sum normalization methods.Results :E … More xposure to CON crystals altered the expression of some of the genes reported previously. Furthermore, novel genes were also identified. The genes for 35 proteins were altered in the expression level by more than 2-fold : annexin V, p53-binding mouse double minute 2 homolog, osteopontin, fibronectin, steroidogenic acute regulatory protein, clusterin, coagulation factor II (thrombin) receptor, mitogen-activated protein kinase 3, cathepsin B, cathepsin L, plasminogen activator inhibitor 1, cytokeratin 8, vimentin, hydrophobic surfactant associated protein C, inhibitor of DNA binding 1, 7-dehydrocholesterol reductase, cytochrome P-45O 2C23, myeloid cell differentiation protein 1, high mobility group protein 2, 5-hydroxytryptamine receptor 5B receptor, cytochrome c expressed in somatic tissues, connective tissue growth factor, tissue inhibitor of metalloproteinase 1, V(D)J recombination activating protein 1, Tclone15, thymosin beta 10, junD proto-oncogene, inhibitor of DNA binding 2, inhibitor of DNA-binding protein 3, G1/S-specific cyclin D1, collagen, insulin-like growth factor binding protein 2, insulin-like growth factor-binding protein 6, profilin 1 and metallothionein 1. Twenty genes had altered expression after a 60-minute exposure to COM crystals. Thirteen genes were up-regulated and seven genes were down-regulated. Nineteen genes had altered expression after a 120-minute exposure to COM crystals. Nine genes were up-regulated and ten genes were down-regulated.Conclusions :We performed a large-scale analysis of gene expression in renal epithelial cells exposed to COM crystals, and identified the genes differentially regulated. cDNA macroarray is a useful tool for evaluating gene expression in urolithiasis research. Less

目的：肾结石的形成是一个复杂的过程，许多基因参与了这个级联反应。一水草酸钙晶体是肾结石中最常见的晶体，其与肾上皮细胞的结合和内化可能是导致肾结石形成的关键步骤。暴露于CON晶体会改变多种基因的表达，但以往对基因表达的研究通常有限。为了更详细地了解基因表达，我们使用cDNA macroarray检测了暴露于CON晶体的肾上皮细胞的基因表达谱。材料与方法：将NRK-52E细胞暴露于CON晶体中60和120分钟。分离Poly (A)^+ RNA并转化为^<32> p标记的第一链cDNA，然后将cDNA探针杂交到膜上。扫描杂交图像，量化信号强度。采用全局和归一化方法分析1176个基因的mRNA表达。结果：更多的接触CON晶体改变了先前报道的一些基因的表达。此外，还发现了新的基因。35个蛋白基因表达水平改变2倍以上；膜联蛋白V、p53结合小鼠双分钟2同源物、骨桥蛋白、纤维连接蛋白、甾体性急性调节蛋白、聚簇蛋白、凝血因子II（凝血酶）受体、丝裂原活化蛋白激酶3、组织蛋白酶B、组织蛋白酶L、纤溶酶原激活物抑制剂1、细胞角蛋白8、纤溶蛋白8、疏水表面活性剂相关蛋白C、DNA结合抑制剂1,7 -脱氢胆固醇还原酶、细胞色素P-45O 2C23、髓细胞分化蛋白1、高迁移率组蛋白2、5-羟色胺受体5B受体、体细胞组织表达的细胞色素c、结缔组织生长因子、组织金属蛋白酶抑制剂1、V(D)J重组激活蛋白1、Tclone15、胸腺素β 10、junD原癌基因、DNA结合蛋白2抑制剂、DNA结合蛋白3抑制剂、G1/ s特异性细胞周期蛋白D1、胶原蛋白、胰岛素样生长因子结合蛋白2、胰岛素样生长因子结合蛋白6、profilin 1和金属硫蛋白1。暴露于COM晶体60分钟后，20个基因的表达发生了变化。13个基因上调，7个基因下调。在暴露于COM晶体120分钟后，19个基因的表达发生了变化。9个基因上调，10个基因下调。结论：我们对暴露于COM晶体的肾上皮细胞的基因表达进行了大规模分析，并确定了差异调控的基因。在尿石症研究中，cDNA大阵列技术是评价基因表达的有效工具。少

项目成果

宮澤 克人: "尿路結石に対するアルコールの影響について"日本尿路結石症学会誌. 2. 92-95 (2003)

Katsuto Miyazawa：“酒精对尿路结石的影响”日本尿路结石病学会杂志 2. 92-95 (2003)。

宮澤 克人: "CaOx結晶による腎尿細管細胞の遺伝子発現について-DNAマクロアレイによる解析"日本尿路結石症学会誌. 1・1. 114-116 (2002)

Katsuto Miyazawa：“CaOx 晶体诱导的肾小管细胞中的基因表达 - 使用 DNA 宏阵列进行分析”日本尿石​​症学会杂志 1・1（2002 年）。

KINUE AIHARA: "Expression of urolithiasis related protein on human renal epithelial cells"Japanese Society on Urolithiasis Research. 11. 183-186 (2001)

KINUE AIHARA：“尿石症相关蛋白在人肾上皮细胞上的表达”日本尿石​​症研究会。

MANABU T MORTYAMA: "Localization of superoxide in oxalate-assosiated injury to HK-2 cells"Japanese Society on Urolithiasis Research. 11. 187-190 (2001)

MANABU T MORTYAMA：“草酸盐相关 HK-2 细胞损伤中超氧化物的定位”日本尿石​​病研究学会。

宮澤 克人: "尿路結石と飲水および飲料物について"泌尿器科紀要. (印刷中).

Katsuto Miyazawa：“关于尿路结石和饮用水和饮料”泌尿外科通报（正在出版）。