[1]王吉利,万雷,张志海,等.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[J].中医正骨,2016,28(09):13-18.
　WANG Jili,WAN Lei,ZHANG Zhihai,et al.Effect of suppression of Dickkopf-1 and Sclerostin expression on bone metabolism regulatory protein expression levels in human osteosarcoma MG63 cells[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(09):13-18.
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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()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第28卷
期数:
2016年09期
页码:
13-18
栏目:
基础研究
出版日期:
2016-09-20

文章信息/Info

Title:
Effect of suppression of Dickkopf-1 and Sclerostin expression on bone metabolism regulatory protein expression levels in human osteosarcoma MG63 cells
作者:
王吉利1; 万雷1; 张志海1; 黄宏兴1; 黄红2; 肖本浩1; 魏合伟1; 曾国勇3
1.广州中医药大学第三附属医院,广东 广州 510240; 2.广州中医药大学,广东 广州 510006; 3.广东省茂名市中医院,广东 茂名 525000
Author(s):
WANG Jili1; WAN Lei1; ZHANG Zhihai1; HUANG Hongxing1; HUANG Hong2; XIAO Benhao1; WEI Hewei1; ZENG Guoyong3
1.The Third Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine,Guangzhou 510240,Guangdong,China2.Guangzhou University of Traditional Chinese Medicine,Guangzhou 510006,Guangdong,China3.Maoming Hospital of Traditional Chinese Medici
关键词:
骨质疏松;  基因沉默;  Dickkopf-1;  Sclerostin;  腺病毒;  MG63细胞
Keywords:
osteoporosis;  gene silencing;  Dickkopf-1;  Serostin;  adenovirus;  MG63 cells
摘要:
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-----------------------------------------------
Abstract:
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 recombinant 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)respectively.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=0.196).The expression of OPG was higher in Sclerostin group and Dickkopf-1-Sclerostin group compared to Scr group(P=0.010,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=0.012,P=0.010,P=0.000).There was no statistical differences in the expression of Lrp-5 between Dickkopf-1 group and Sclerostin group(P=0.119).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=0.010,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=0.010,P=0.000,P=0.010).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 compared 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=0.170).The expression of OPN was lower in Dickkopf-1 group compared to Dickkopf-1-Sclerostin group(P=0.000).The expression of TNF-α was 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 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-α between Sclerostin group and Dickkopf-1-Sclerostin group(P=0.125).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 expression levels of bone metabolism regulatory proteins are influenced more by suppression of Sclerostin expression than suppression of Dickkopf-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.

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备注/Memo

备注/Memo:
2016-07-14收稿 2016-08-09修回
基金项目:国家自然科学基金项目(81302991,81373653); 广东省自然科学基金项目(S2013040016828,2014A030310127); 广东省科技计划项目(2013B021800058); 广州中医药大学青年英才项目(QNYC20120119); 2014广东省“优青计划”项目(yq2014041)
万雷 E-mail:gzwl802@163.com

更新日期/Last Update: 1900-01-01