骨桥蛋白在高钙促进大鼠肾上皮细胞与草酸钙晶体黏附中的作用  

Effects of osteopontin on hypercalciuria-induced attachment of calcium oxalate crystals to rat renal epithelial cells

作　　者：席启林[1] 刘喆[2] 侯建全[1] 欧阳骏[1] 浦金贤[1] 刘安定[3] 王少刚[4] 叶章群[4] 

机构地区：[1]苏州大学附属第一医院泌尿外科,215006 [2]江苏省无锡市第二人民医院 [3]华中科技大学同济医学院附属同济医院实验医学中心 [4]华中科技大学同济医学院附属同济医院泌尿外科

出　　处：《中华实验外科杂志》2014年第8期1762-1765,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81170651）

摘　　要：目的 探讨骨桥蛋白（OPN）在高钙尿致含钙肾结石形成中的作用及机制.方法 根据成人及大鼠高钙尿诊断标准,钙干预浓度分别为0.1、0.3、0.6 g/L,作用于大鼠肾上皮细胞（NRK）,检测其对NRK细胞增殖、分泌OPN及OPN基因表达的影响;构建有效沉默OPN基因表达的慢病毒载体[OPN-RNA干扰（RNAi）],感染NRK细胞后应用高钙刺激,检测细胞黏附的一水草酸钙晶体量的变化.结果 0.3、0.6 g/L钙刺激NRK细胞6、24、48 h后,3个时间点检测NRK细胞增殖均较空白对照组明显减慢,差异有统计学意义（P＜0.05）;3个浓度钙刺激3、6、24h后OPN分泌量均较空白对照组显著增加,差异有统计学意义（P＜0.05）;3个浓度干预3h和6h两个时间点NRK细胞OPN基因mRNA和蛋白水平均明显升高,与空白对照组比较差异均有统计学意义（P＜0.05）;成功构建OPN-RNAi慢病毒载体,病毒滴度为6×108 TU/ml.OPN-RNAi组、空病毒载体组和空白对照组分别用0.6 g/L钙干预24 h后细胞黏附的一水草酸钙含量（以钙浓度表示,mg/L）分别为19.25±3.71、27.22±3.82和31.91±1.15;OPN-RNAi组细胞晶体黏附量较空病毒载体组和空白对照组均显著减少,差异均有统计学意义（P＜0.05）.结论 OPN可能介导高钙促进NRK细胞与一水草酸钙晶体的黏附.Objective To address the effects of osteopontin （OPN） on hypercalciuria contributing to the kidney stone formation.Methods The normal rat kidney epithelial cell line NRK cells were divided into three groups,which were cultured in the medium containing calcium of 0.1,0.3 and 0.6 g/L,respectively.The cell proliferation,the content of OPN in the cell culture supernatants and the level of OPN gene expression at 3,6 and 24 h were examined.The lentivirus vector targeting OPN was constructed.The NRK cells were infected with the lentivirus and then cultured in the medium containing calcium of 0.6 g/L for 24 h.Eventually,The inhibitory effects of silencing OPN gene on the adhesion of calcium oxalate crystals to NRK cells were investigated.Results High level calcium could significantly suppress the proliferation of NRK cells and stimulate secretion of OPN as compared with the blank control group （P ＜ 0.05）.The expression of OPN mRNA and protein was also significantly increased （P ＜ 0.05）.Calcium concentrations of the lysate of infected NRK cells in the three groups after calcium chloride stimulation were （19.25 ±3.71）,（27.22 ±3.82） and （31.91 ± 1.15） mg/L,respectively.The silencing of OPN gene expression could significantly inhibit the abilities of attachment of calcium oxalate crystals to NRK cells as compared with negative lentivirus vector and blank control group,respectively （P ＜ 0.05）.Conclusion High level calcium could induce the adhesion of calcium oxalate crystals to NRK cells probably mediated by OPN.

关 键 词：高钙尿 骨桥蛋白 肾上皮细胞 

分 类 号：R691.4[医药卫生—泌尿科学]