ERK信号通路调节金属离子诱导成骨细胞RANKL表达的实验研究

目的探讨小鼠成骨细胞暴露于金属钴、铬离子条件下对RANKL表达的影响及ERK信号通路在这一过程中作用,寻找防治假体周围骨溶解的方法。方法体外培养成骨细胞,细胞密度为1×105个/ml。根据培养液的不同,实验分三组：对照组给予生理盐水（A组）,实验组1给予钴、铬离子干预（B组）,实验组2给予钴、铬离子＋ERK信号通路抑制剂PD98059干预（C组）,共培养24h、48h后,用RT-PCR和ELISA法对RANKL进行基因水平和分泌蛋白水平检测。结果RT-PCR结果显示：三组各个时间点均可见RANKL的表达,B、C组RANKL基因表达较A组均增加,C组较B组RANKL基因表达明显下降,差异均有统计学意义（P〈0.01）。ELISA结果显示：24h后B、C组成骨细胞RANKL蛋白的分泌量分别是A组的61.6倍、44.4倍;48h后B、C组成骨细胞RANKL蛋白的分泌量分别是A组的13.8倍、10.5倍,差异均有统计学意义（P〈0.01）。C组在培养24h、48h后RANKL的表达与B组相比分别下降约27.6%、23.6%,差异均有统计学意义（P〈0.01）。结论金属离子可刺激成骨细胞RANKLmRNA的表达并促进RANKL蛋白的分泌;ERK信号通路抑制剂PD98059可一定程度抑制RANKL的表达,对减少假体周围骨溶解的发生可能起重要作用。

ERK signaling regulates RANKL expression induced by metal ions in MC3T3E1 murine calvarial preosteoblastic cells

Objective To investigate the effects on the expression of RANKL from osteoblasts（MC3T3E1）exposed to cobalt（Co2＋）and chromium（Cr3＋）ions and the effect of ERK-MAPK in the above process and to explore the methods of preventing the periprosthetic osteolysis.Methods Osteoblasts were cultured in vitro,in a density of 1×105 cells/ml,and divided into three groups according to the used culture solution.In control group（group A）,osteoblasts were cultured with normal saline;in experimental group one（group B）,osteoblasts were cultured with cobalt（Co2＋）and chromium（Cr3＋）ions solution;in experimental group two（group C）,osteoblasts were cultured with Co2＋,Cr3＋ solution and inhibitor of ERK signaling（PD98059）.The RT-PCR and ELISA methods were applied to detect the expression of RANKL mRNA and protein at 24 and 48 h after co-culture respectively.Results RT-PCR revealed that the gene expression of RANKL could be detected in three groups at 24 and 48 h after culture.The expression level of RANKL mRNA in groups B and C was higher than that in control A,and that in group C was decreased obviously as compared with that in group B（all P〈0.01）.ELISA revealed that the expression of RANKL in experimental groups B and C was increased obviously as compared with that in control group,and as compared with group A,the RANKL was increased to 61.6-fold,44.4-fold respectively at 24 h and 13.8-fold,10.5-fold respectively at 48 h（all P〈0.01）.After culture for 24 and 48 h,the expression of RANKL in group C was decreased obviously by 27.6% and 23.6% as compared with that in group B（P〈0.01）.Conclusion Co2＋ and Cr3＋ ions could stimulate the expression of RANKL mRNA and the secretion of RANKL protein from osteoblasts.PD98059 could restrain the expression of RANKL at a certain degree and decrease the occurrence of the periprosthetic osteolysis.