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A Study for Elucidation of Functional Mechanism of High Molecular Weight Substances in Urine on Calcium Oxalate Stone Formation

阐明尿液中高分子物质对草酸钙结石形成作用机制的研究

基本信息

批准号：
12307032
负责人：
YACHIKU Sunao
金额：
$ 25.45万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

1. Study of crystal-cell interactions: An experimental model of calcium oxalate (CaOx) stones, suitable for investigation of crystal-cell interactions, has been established. It was confirmed that the phosphatidylserine exposure on the outer leaflet of the cell membrane of renal collecting duct cells could be evaluated quantitatively by using this model. Increased phosphatidylserine expressing cells and activation of the caspase family (intracellular signal transduction systems for apoptosis) were noted with increasing urinary oxalate excretion and calcium oxalate crystals. 2. Purification of stone-related proteins and their molecular biological analysis: Osteopontin (OPN) and calprotectin were purified. The adhesion of CaOx crystals to MDCK cells was promoted by OPN, while it was suppressed by thrombin and antisense oligonucleotide. It seems likely that OPN-DNA polymorphism in patients with urinary tract stones is involved in mutation of the enhancer regions that are related to synthes … More is of OPN protein. 3. OPN antisense gene transfer: The introduction of OPN antisense gene to renal tubular cells suppressed OPN expression and CaOx stone formation in the kidneys. The addition of OPN and vitronectin to renal tubular cells in culture elevated the intracellular expression of OPN and promoted the phosphorylation of focal adhesion kinase. Catechin suppressed the formation of CaOx crystals through inhibiting apoptosis induced by oxidative stress. 4. Study of urinary tract stone inhibitors using antisense gene: Human syndecan-1-expressing renal tubular cells were established. Heparan sulfate (HS) and HS proteoglycan were found to suppress the cellular injury by oxalate exposure and the adhesion of CaOx crystals to the cell surface. It was suggested that HS prevented the cell injury by serving as a charge barrier that inhibited the influx of oxalate ions into the cells. 5. Effects to stone formation by controlling the expression of stone-related protein mRNA: Increased expression of renal prothrombin F-1 (RPTF-1) mRNA in rat kidneys with CaOx was confirmed. In the macro-array analysis, it was noted that RPTF-1 protein, OPN gene, and the multiple genes encoding inflammatory systems were increased after CaOx crystal exposure to the renal tubular cells. Less

1.晶体细胞相互作用的研究：草酸钙（CaOx）结石的实验模型，适合于研究晶体细胞的相互作用，已经建立。结果表明，该模型可定量评价肾集合管细胞膜外叶磷脂酰丝氨酸的暴露量。随着尿草酸盐排泄和草酸钙结晶的增加，观察到表达磷脂酰丝氨酸的细胞增加和半胱天冬酶家族（细胞凋亡的细胞内信号转导系统）的激活。2.结石相关蛋白的纯化及其分子生物学分析：纯化骨桥蛋白（OPN）和钙卫蛋白。OPN可促进CaOx晶体与MDCK细胞的粘附，而凝血酶和反义寡核苷酸则可抑制CaOx晶体与MDCK细胞的粘附。尿路结石患者的OPN-DNA多态性可能与合成酶相关的增强子区域的突变有关。 ...更多信息是OPN蛋白。3. OPN反义基因转移：将OPN反义基因导入肾小管细胞可抑制OPN表达和肾内CaOx结石形成。在培养的肾小管细胞中加入OPN和Vitronectin可提高细胞内OPN的表达，并促进黏着斑激酶的磷酸化。儿茶素通过抑制氧化应激诱导的细胞凋亡抑制CaOx晶体的形成。4.反义基因抑制尿路结石的研究：建立表达人syndecan-1的肾小管细胞。硫酸乙酰肝素（HS）和HS蛋白聚糖被发现可以抑制草酸暴露引起的细胞损伤和CaOx晶体粘附到细胞表面。结果表明，HS可作为一种电荷屏障，抑制草酸根离子进入细胞，从而防止细胞损伤。5.通过控制结石相关蛋白mRNA的表达对结石形成的影响：证实了CaOx大鼠肾脏中肾凝血酶原F-1（RPTF-1）mRNA的表达增加。在宏阵列分析中，注意到在CaOx晶体暴露于肾小管细胞后，RPTF-1蛋白、OPN基因和编码炎症系统的多个基因增加。少