DKK1基因沉默的人多发性骨髓瘤细胞培养上清对小鼠前成骨细胞成骨分化的影响

摘要

AIM:To investigate the effect of Dickkopt-1 (DKK1) gene on myeloma bone disease.METHODS:Lentivirus was packed with pLenti6/V5-GW/DKK-1-miR plasmids and lentivirus packaging plasmids,and then transfected into human multiple mydoma cell line U266.An in vitro osteogenic experiment was performed and the mouse MC3T3-E1 preosteoblasts were divided into blank control group,bone morphogenetic protein 2 (BMP-2) induction group,U266 supernatant group,DKK1 RNAi U266 supematant group and negative RNAi U266 supematant group.All supematants added to the cells were at the concentration of 30％.After culture for 12 d,the effects of different U266 cell supernatants on osteogenic differentiation of MC3T3-E1 cells were investigated by alizarin red staining.The mRNA expression of osteocalcin (OC) was detected by real-time PCR.RESULTS:Compared with blank control group,the mRNA expression of OC in BMP-2 induction group increased significantly.Compared with BMP-2 induction group,the mRNA expression of OC and the number of calcium nodule in U266 supernatant group were decreased.Compared with U266 supematant group,the mRNA expression of OC and the number of calcium nodule in DKK1 RNAi supematant group were significantly elevated.In negative RNAi U266 supernatant group,0C mRNA and the calcium nodule did not change significantly.CONCLUSION:After DKK1gene silencing in U266 cells by RNAi,the inhibition of osteogenic diferentiafion of mouse preosteoblasts significantly decreases.%目的:初步探讨一种与胚胎发生及肿瘤发展密切相关的糖蛋白Dickkopf-1(DKK1)在骨髓瘤骨病(myeloma bone disease,MBD)发病中的作用.方法:前期实验构建的质粒pLenti6/V5-GW/DKK-1-miR,与慢病毒包装质粒一起包装成慢病毒,感染人骨髓瘤U266细胞,建立DKK1沉默的U266细胞.小鼠前成骨细胞MC3T3-E1体外成骨诱导实验分组:空白对照组、诱导分化组、30％ U266上清干预组、30％ DKK1 RNAi U266上清干预组和30％无关序列RNAi U266上清干预组；诱导培养12 d后,通过茜素红染色检测钙结节数目；real-time PCR检测骨钙素(osteocalcin,OC) mRNA的表达变化.结果:成骨诱导体系中,与空白组比较,诱导组OC mRNA有明显升高,差异有统计学意义(P＜0.01).与诱导组比较,30％ U266上清干预组OC mRNA表达明显减少(P＜0.01).与30％U266上清干预组比较,30％ DKK1 RNAi U266上清干预组OC mRNA表达明显增多(P＜0.05),30％无关序列RNAi U266上清干预组OC mRNA差异无统计学意义.与诱导组比较,30％ U266上清干预组钙结节计数明显减少(P＜0.01).与30％ U266上清干预组比较,30％ DKK1 RNAi U266上清干预组钙结节计数明显增多(P＜0.05),30％无关序列RNAi U266上清干预组钙结节计数差异无统计学意义(P＞0.05).结论:U266细胞中DKK1基因沉默后,其培养上清抑制小鼠前成骨细胞MC3T3-E1成骨分化的能力减弱.