摘要
目的探讨槲皮素对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)增殖和成骨分化以及对特异AT序列结合蛋白2(SATB2)基因表达的影响。方法噻唑蓝(MTT)法检测不同浓度槲皮素对hBMSCs增殖活性的影响,碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测ALP活性,实时荧光定量PCR(qRT-PCR)检测成骨分化生物标志物Runt相关转录因子2(Runx2)、骨钙素(BGLAP)和分泌磷酸蛋白1(SPP1)mRNA的表达,qRT-PCR和蛋白免疫印迹(Western blot)检测SATB2 mRNA和蛋白表达变化,在hBMSCs转染SATB2特异性siRNA或SATB2过表达载体质粒,分析敲低或过表达SATB2对槲皮素干预的hBMSCs增殖和成骨分化的影响。结果在一定范围内槲皮素呈浓度依赖性促进hBMSCs增殖,5μmol/L时促进细胞增殖效果最佳,选择5μmol/L的槲皮素用于后续研究。5μmol/L的槲皮素能够有效促进ALP活性以及Runx2、BGLAP和SPP1 mRNA的表达,并且能够上调SATB2 mRNA和蛋白表达。敲低SATB2能够阻碍槲皮素对hBMSCs增殖和成骨分化的促进作用,而过表达SATB2能够增强槲皮素对hBMSCs增殖和成骨分化的促进作用。结论槲皮素可通过上调SATB2基因的表达促进hBMSCs增殖和成骨分化。
Objective To investigate the effect of quercetin on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)and on the expression of specific AT sequence binding protein 2(SATB2).Methods Thiazole blue(MTT)method was used to detect the effect of different concentrations of quercetin on the proliferation activity of hBMSCs.Alkaline phosphatase(ALP)kit was used to detect ALP activity.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the osteogenic differentiation biomarkers runt-related transcription factor 2(Runx2),osteocalcin(BGLAP),and secreted phosphoprotein 1(SPP1)mRNA expression.qRT-PCR and Western blotting were used to detect SATB2 mRNA and protein expression changes.Specific siRNA or SATB2 overexpression vector plasmids were transfected in hBMSCs to analyze the effect of knockdown or overexpression of SATB2 on the proliferation and osteogenic differentiation of hBMSCs intervened by quercetin.Results Quercetin promoted the proliferation of hBMSCs in a concentration-dependent manner within a certain range,and the best effect of promoting cell proliferation was at 5μmol/L.Quercetin at 5μmol/L was selected for follow-up research.Quercetin at 5μmol/L effectively promoted ALP activity and the expression of Runx2,BGLAP,and SPP1 mRNA,and up-regulated SATB2 mRNA and protein expression.Knockdown of SATB2 inhibited the promotion of quercetin on the proliferation and osteogenic differentiation of hBMSCs,while overexpression of SATB2 enhanced the promotion of quercetin on the proliferation and osteogenic differentiation of hBMSCs.Conclusion Quercetin promotes the proliferation and osteogenic differentiation of hBMSCs by up-regulating the expression of SATB2 gene.
作者
张波
胡凌云
苟林
贾敏
李永宁
罗栩伟
李伟
ZHANG Bo;HU Lingyun;GOU Lin;JIA Min;LI Yongning;LUO Xuwei;LI Wei(Department of Orthopedics,Nanchong Central Hospital,Nanchong 637000,China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2022年第12期1765-1769,1776,共6页
Chinese Journal of Osteoporosis
基金
南充市科技战略合作专项任务(18SXHZ0374)。
关键词
槲皮素
SATB2基因
骨髓间充质干细胞
增殖
成骨分化
quercetin
SATB2 gene
bone marrow mesenchymal stem cells
proliferation
osteogenic differentiation
