高钙离子诱导人肾小管上皮细胞表达M CP-1和HM GB1

摘要

目的:观察体外培养的人肾小管上皮细胞在高钙离子环境下细胞损伤及炎性因子MCP-1和HMGB1的表达.方法:体外培养人肾小管上皮细胞(HK-2细胞),使用CaCl2配制成不同钙离子浓度的溶液作为刺激剂,实验分为正常对照组(正常培养液)、Ca I组(5 mmol/L Ca2+液)、CaⅡ组(10 mmol/L Ca2+液)和CaⅢ组(15 mmol/L Ca2+液).各组细胞分别培养至3 h、6 h、9 h时,采用MTT法检测细胞活力.培养6 h时,采用4′,6-二脒基-2-苯基吲哚(DAPI)进行细胞凋亡染色,观察各组细胞凋亡情况;同时收集细胞培养上清液,采用ELISA法分别检测各组细胞上清液过氧化氢(H2O2)和8-异前列烷(8-IP)浓度,微量酶标仪法检测各组细胞上清液乳酸脱氢酶(LDH)活性.此外,培养6 h时收集各组细胞,采用real-time PCR法检测细胞MCP-1和HMGB1的mRNA表达情况;并收集各组细胞上清液,采用ELISA法检测其MCP-1和HMGB1的含量.结果:随着细胞培养液中钙离子浓度的增加,细胞活力抑制率和细胞凋亡明显增加.LDH活性、细胞上清液H2 O2的浓度和8-IP的含量在Ca I组、Ca II组和Ca III组均较正常对照组高(P<0.05).real-time PCR的结果显示,与对照组比较,3个钙离子诱导组细胞MCP-1和HMGB1的mRNA表达水平升高,差异有统计学显著性(P<0.05).此外,3个钙离子诱导组细胞上清液MCP-1和HMGB1含量均较正常组高(P<0.05).结论:本研究结果提示,高钙离子可诱导人肾小管上皮细胞出现氧化应激损伤,同时细胞高表达MCP-1和HMGB1.由MCP-1和HMGB1引发的炎症反应可能是高钙尿参与结石形成的重要机制.%AIM:To investigate the effect of high level of calcium on the expression of inflammatory cytokines MCP-1 and HMGB1 in human kidney epithelial HK-2 cells.METHODS:Cultured HK-2 cells were divided into control group (normal media), CaⅠgroup (5 mmol/L Ca2+media), CaⅡgroup (10 mmol/L Ca2+media) and CaⅢgroup (15 mmol/L Ca2+media) .The cells were incubated with or without additives for 3 h, 6 h and 9 h, and the cell activity was measured by MTT assay.At 6 h, DAPI staining was used to observe the cell apoptosis.LDL activity, H2 O2 content and 8-isoprostane (8-IP) concentration in the media were determined by microplate reader microfiltration and ELISA.The mRNA expression of MCP-1 and HMGB1 in the cells were determined by real-time PCR, and the protein levels of MCP-1 and HMGB1 in the media were measured by ELISA.RESULTS:The results of MTT assay showed that the inhibition rate of cell activity increased significantly with the increase in calcium concentration in the media.The results of DAPI staining showed that the apoptotic cells increased with the increase in calcium concentration in the media.The LDL activity, H2 O2 content and 8-IP concentration in the media all increased in accordance with the increased concentrations of calcium supple-mented.Compared with control group, the mRNA expression of MCP-1 and HMGB1 in the HK-2 cells increased signifi-cantly in the 3 calcium supplemented groups (P<0.05).The protein levels of MCP-1 and HMGB1 in the media of Ca I, Ca II and CaⅢgroups were significantly increased compared with control group (P<0.05).CONCLUSION:The pro-duction of MCP-1 and HMGB1 in the HK-2 cells exposed to high level of calcium is significantly increased, in accordance with the oxidative cell injury.