抑制Dickkopf-1和Sclerostin表达对人成骨肉瘤细胞MG63骨代谢调节相关蛋白表达水平的影响

Effect of suppression of Dickkopf- 1 and Sclerostin expression on bone metabolism regulatory protein expression levels in human osteosarcoma MG63 cells

目的:观察抑制Dickkopf-1和Sclerostin表达对人成骨肉瘤细胞MG63骨代谢调节相关蛋白表达水平的影响。方法:培养MG63细胞,构建沉默Dickkopf-1重组腺病毒载体和沉默Sclerostin重组腺病毒载体。将培养好的MG63细胞(每孔2×105个)分为4组,Scr组以Scr腺病毒转染、Dickkopf-1组以沉默Dickkopf-1重组腺病毒载体转染、Sclerostin组以沉默Sclerostin重组腺病毒载体转染、Dickkopf-1+Sclerostin组以沉默Dickkopf-1重组腺病毒载体和沉默Sclerostin重组腺病毒载体共同转染。各组MG63细胞转染重组腺病毒48 h后以Western Blot法测定各组细胞中骨保护素(osteoprotegerin,OPG)、低密度脂蛋白受体相关蛋白-5(low density lipoprotein receptor-related protein-5,Lrp-5)、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)、成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)、Runt相关转录因子-2(Runt-related transcription factor-2,Runx-2)、结缔组织生长因子(connective tissue growth factor,CTGF)、骨桥蛋白(osteopontin,OPN)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)等骨代谢调节相关蛋白的表达水平。结果:Dickkopf-1组的OPG表达量与Scr组、Sclerostin组比较,组间差异均无统计学意义(P=0.050,P=0.196);Sclerostin组、Dickkopf-1+Sclerostin组的OPG表达量均高于Scr组(P=0.010,P=0.000);Dickkopf-1组和Sclerostin组的OPG表达量均低于Dickkopf-1+Sclerostin组(P=0.000,P=0.000)。Dickkopf-1组、Sclerostin组、Dickkopf-1+Sclerostin组的Lrp-5表达量均高于Scr组(P=0.012,P=0.010,P=0.000);Dickkopf-1组的Lrp-5表达量与Sclerostin组比较,差异无统计学意义(P=0.119);Dickkopf-1组和Sclerostin组的Lrp-5表达量均低于Dickkopf-1+Sclerostin组(P=0.000,P=0.000)。Dickkopf-1组的BMP-2表达量与Scr组比较,差异无统计学意义(P=0.185);Dickkopf-1组的BMP-2表达量低于Sclerostin组(P=0.037);Dickkopf-1组和Sclerostin组的BMP-2表达量均低于Dickkopf-1+Sclerostin组(P=0.000,P=0.000)。Dickkopf-1组、Sclerostin组、Dickkopf-1+Sclerostin组的FGF-2表达量均高于Scr组(P=0.010,P=0.000,P=0.000);Dickkopf-1组的FGF-2表达量低于Sclerostin组和Dickkopf-1+Sclerostin组(P=0.000,P=0.000);Sclerostin组的FGF-2表达量低于Dickkopf-1+Sclerostin组(P=0.000)。Dickkopf-1组、Sclerostin组、Dickkopf-1+Sclerostin组的Runx-2表达量均高于Scr组(P=0.000,P=0.000,P=0.000);Dickkopf-1组的Runx-2表达量低于Sclerostin组和Dickkopf-1+Sclerostin组(P=0.000,P=0.000);Sclerostin组的Runx-2表达量低于Dickkopf-1+Sclerostin组(P=0.000)。Dickkopf-1组、Sclerostin组、Dickkopf-1+Sclerostin组的CTGF表达量均高于Scr组(P=0.010,P=0.000,P=0.010);Dickkopf-1组的CTGF表达量与Sclerostin组比较,差异无统计学意义(P=0.080);Dickkopf-1组和Sclerostin组的CTGF表达量均低于Dickkopf-1+Sclerostin组(P=0.000,P=0.004)。Dickkopf-1组、Sclerostin组、Dickkopf-1+Sclerostin组的OPN表达量均高于Scr组(P=0.002,P=0.000,P=0.000);Dickkopf-1组和Dickkopf-1+Sclerostin组的OPN表达量与Sclerostin组比较,差异均无统计学意义(P=0.050,P=0.170);Dickkopf-1组的OPN表达量低于Dickkopf-1+Sclerostin组(P=0.000)。Dickkopf-1组、Sclerostin组、Dickkopf-1+Sclerostin组的TNF-α表达量均低于Scr组(P=0.000,P=0.000,P=0.000);Dickkopf-1组的TNF-α表达量高于Sclerostin组和Dickkopf-1+Sclerostin组(P=0.000,P=0.000);Sclerostin组和Dickkopf-1+Sclerostin组的TNF-α表达量比较,差异无统计学意义(P=0.125)。结论:抑制MG63细胞Dickkopf-1和Sclerostin表达,均能上调具有骨形成和双向调节作用的骨代谢调节相关蛋白表达水平、下调具有骨吸收作用的骨代谢调节相关蛋白表达水平,其中抑制Sclerostin表达对骨代谢调节相关蛋白表达水平的影响强于抑制Dickkopf-1表达,而且抑制Dickkopf-1和Sclerostin表达在调节骨代谢调节相关蛋白表达水平方面具有协同作用。

Objective：To observe the effect of suppression of Dickkopf-1 and Sclerostin expression on bone metabolism regulatory protein expression levels in human osteosarcoma MG63 cells.Methods：The MG63 cells were cultured and Dickkopf-1 silencing recombi-nant adenovirus vectors and Sclerostin silencing recombinant adenovirus vectors were constructed.The MG63 cells were divided into 4 groups,two hundred thousand cells in one hole,and were transfected with Scr adenovirus（Scr group）,Dickkopf-1 silencing recombinant adenovirus vectors（Dickkopf-1 group）,Sclerostin silencing recombinant adenovirus vectors（Sclerostin group）and Dickkopf-1 silencing recombinant adenovirus vectors combined with Sclerostin silencing recombinant adenovirus vectors（Dickkopf-1 -Sclerostin group）respec-tively.At 48 hours after recombinant adenovirus transfection,Western Blot was used to measure the expression levels of bone metabolism regulatory protein,including osteoprotegerin（OPG）,low density lipoprotein receptor-related protein -5 （Lrp -5 ）,bone morphogenetic protein-2（BMP-2）,fibroblast growth factor-2（FGF-2）,Runt-related transcription factor-2（Runx-2）,connective tissue growth factor（CTGF）,osteopontin（OPN）and tumor necrosis factor-α（TNF-α）.Results：There was no statistical difference in the expression of OPG between Dickkopf-1 group and Scr group and between Dickkopf-1 group and Sclerostin group（P=0.050,P=0.1 96）.The expres-sion of OPG was higher in Sclerostin group and Dickkopf-1 -Sclerostin group compared to Scr group（P=0.01 0,P=0.000）,and was lower in Dickkopf-1 group and Sclerostin group compared to Dickkopf-1 -Sclerostin group（P=0.000,P=0.000）.The expression of Lrp-5 was higher in Dickkopf-1 group,Sclerostin group and Dickkopf-1 -Sclerostin group compared to Scr group（P=0.01 2,P=0.01 0,P=0.000）.There was no statistical differences in the expression of Lrp-5 between Dickkopf-1 group and Sclerostin group（P=0.1 1 9）.The expression of Lrp-5 was lower in Dickkopf-1 group and Sclerostin group compared to Dickkopf-1 -Sclerostin group（P=0.000,P=0.000）.There was no statistical differences in the expression of BMP-2 between Dickkopf-1 group and Scr group（P=0.185）.The expression of BMP -2 was lower in Dickkopf-1 group compared to Sclerostin group（P =0.037）,and was lower in Dickkopf-1 group and Sclerostin group compared to Dickkopf-1 -Sclerostin group（P=0.000,P=0.000）.The expression of FGF-2 was higher in Dickkopf-1 group,Sclerostin group and Dickkopf-1 -Sclerostin group compared to Scr group（P=0.01 0,P=0.000,P=0.000）,and was lower in Dickkopf-1 group compared to Sclerostin group and Dickkopf-1 -Sclerostin group（P=0.000,P=0.000）. The expression of FGF-2 was lower in Sclerostin group compared to Dickkopf-1 -Sclerostin group（P=0.000）.The expression of Runx-2 was higher in Dickkopf-1 group,Sclerostin group and Dickkopf-1 -Sclerostin group compared to Scr group（P=0.000,P=0.000,P=0.000）.The expression of Runx-2 was lower in Dickkopf-1 group compared to Sclerostin group and Dickkopf-1 -Sclerostin group（P=0.000,P=0.000）,and was lower in Sclerostin group compared to Dickkopf-1 -Sclerostin group（P=0.000）.The expression of CTGF was higher in Dickkopf-1 group,Sclerostin group and Dickkopf-1 -Sclerostin group compared to Scr group（P=0.01 0,P=0.000,P=0.01 0）.There was no statistical differences in the expression of CTGF between Dickkopf-1 group and Sclerostin group（P=0.080）.The expression of CTGF was lower in Dickkopf-1 group and Sclerostin group compared to Dickkopf-1 -Sclerostin group（P=0.000,P=0.004）.The expression of OPN was higher in Dickkopf-1 group,Sclerostin group and Dickkopf-1 -Sclerostin group com-pared to Scr group（P=0.002,P=0.000,P=0.000）.There was no statistical differences in the expression of OPN between Dickkopf-1 group and Sclerostin group and between Dickkopf-1 -Sclerostin group and Sclerostin group（P=0.050,P=0.1 70）.The expression of OPN was lower in Dickkopf-1 group compared to Dickkopf-1 -Sclerostin group（P=0.000）.The expression of TNF-αwas lower in Dickkopf-1 group,Sclerostin group and Dickkopf-1 -Sclerostin group compared to Scr group（P=0.000,P=0.000,P=0.000）,and＆amp;nbsp;was higher in Dickkopf-1 group compared to Sclerostin group and Dickkopf-1 -Sclerostin group（P=0.000,P=0.000）.There was no statistical differences in the expression of TNF-αbetween Sclerostin group and Dickkopf-1 -Sclerostin group（P=0.1 25）.Conclusion：By suppressing the expression of Dickkopf-1 and Sclerostin in MG63 cells,the expression levels of bone metabolism regulatory proteins which has accelerating effects on bone formation and which has bidirectional regulatory effects on bone metabolism can be upregulated,and the expression levels of bone metabolism regulatory proteins which has accelerating effects on bone resorption can be downregulated.The ex-pression levels of bone metabolism regulatory proteins are influenced more by suppression of Sclerostin expression than suppression of Dick-kopf-1 expression,and the suppression of expression of Dickkopf-1 and Sclerostin has a synergistic effect on regulating the expression levels of bone metabolism regulatory proteins.