羟基磷灰石对人肾小管上皮细胞骨桥蛋白表达的影响及机制探讨

摘要

目的 观察羟基磷灰石(HAP)对人肾小管上皮细胞株HK-2细胞骨桥蛋白(OPN)表达的影响,探讨HAP在肾结石形成中的作用机制.方法 体外培养HK-2细胞,分为正常对照组、HAP1组、HAP2组、HAP3组及HAP4组.正常对照组在正常培养液中培养,HAP1~4组分别在正常培养液中加入200、400、600、800 ng/mL的HAP悬液.各组培养至12、24、48、72 h时,采用CCK-8法检测细胞活力.培养至24 h时,采用RT-qPCR法检测各组细胞中OPN mRNA的表达,采用Western blot法检测各组细胞中OPN蛋白的表达.结果 随着HAP浓度增加,细胞存活率逐渐降低,细胞存活率与HAP的浓度呈负相关(r=-0.566 3,P<0.01).随着HAP作用时间延长,细胞存活率逐渐降低,细胞存活率与HAP作用时间呈负相关(r=-0.796 3,P<0.01).正常对照组、HAP1组、HAP2组、HAP3组、HAP4组OPN mRNA相对表达量分别为1.151±0.029、1.201±0.004、1.269±0.029、1.179±0.022、1.406±0.058,HAP2组、HAP4组OPN mRNA相对表达量均高于正常对照组(P均<0.05).正常对照组、HAP1组、HAP2组、HAP3组、HAP4组OPN蛋白相对表达量分别为0.418±0.010、0.436±0.016、0.512±0.021、0.562±0.016、0.627±0.023,HAP2、HAP3、HAP4组OPN蛋白相对表达量均高于正常对照组(P均<0.05).结论 HAP可使人肾小管上皮细胞出现损伤并上调OPN表达,可能是其参与肾结石形成的机制之一.%Objective To investigate the effect of hydroxyapatite (HAP) on osteopontin (OPN) expression in human renal tubular epithelial cells (HK-2 cells) and to explore the role of HAP in the formation of kidney stones.Methods The cultured HK-2 cells were divided into the control group,HAP1 group,HAP2 group,HAP3 group and HAP4 group.HK-2 cells in the control group were cultured in the normal culture media,from HAP 1 group to HAP 4 group,we respectively added 200,400,600,400 ng/mL HAP suspension to the culture media.After 12,24,48 and 72 h,the cell activity was measured by CCK-8 assay.In addition,when cultured to 24 hours,RT-qPCR and Western blotting were applied to detect the expression levels of OPN mRNA and protein in human renal tubular epithelial cells.Results With the increase of HAP concentration,the cell viability was decreased gradually,and the cell viability was negatively correlated with the concentrations of HAP (r=-0.566 3,P<0.01).With the prolonged time of HAP,the cell viability was decreased and the cell viability was negatively correlated with the time of HAP (r=-0.796 3,P<0.01).The relative expression levels of OPN mRNA in the normal control group,HAP1 group,HAP2 group,HAP3 group and HAP4 group were 1.151±0.029,1.201±0.004,1.269±0.029,1.179±0.022,and 1.406±0.058,respectively.The OPN mRNA expression of the HAP2 group and HAP4 group was higher than that of the normal control group (all P<0.05).The expression levels of OPN protein in the normal control group,HAP1 group,HAP2 group,HAP3 group and HAP4 group were 0.418±0.010,0.436±0.016,0.512±0.021,0.562±0.016 and 0.627±0.023,respectively.The OPN protein expression of the HAP2,HAP3 and HAP4 groups was higher than that of the normal control group (all P<0.05).Conclusion HAP can damage the renal tubular epithelial cells and up-regulate the expression of OPN,which may be one of the mechanisms involved in the formation of kidney stones.