摘要
目的探讨萝卜硫素(Sul)促进骨髓间充质干细胞(BMSCs)向成骨细胞分化的分子机制。方法将BMSCs分为对照组(不做任何处理)、诱导组(诱导成骨分化)、诱导+Sul组(诱导成骨分化并加入40µmol/L Sul);分别向BMSCs转染腺病毒-shRNA-Mock、-shRNA-TET1、-shRNA-TET2、-shRNA-TET3,作为shRNA-Mock组、shRNA-TET1组、shRNA-TET2组、shRNA-TET3组;BMSCs于含成骨分化诱导培养液及添加40µmol/L Sul的细胞培养液中培养,然后转染腺病毒-shRNA-TET1、-shRNA-TET2、-shRNA-TET3、-shRNA-Mock,作为诱导+Sul+shRNA-TET1组、诱导+Sul+shRNA-TET2组、诱导+Sul+shRNA-TET3组、诱导+Sul+shRNA-Mock组。qPCR法和Western blot法测定BMSCs向成骨细胞分化后Runx2 mRNA和蛋白的表达水平。染色质免疫共沉淀(ChIP)实验测定BMSCs向成骨细胞分化后,与Histone H3结合的Runx2启动子区DNA含量。HpaⅡ酶和MspⅠ酶消化联合qPCR法测定BMSCs向成骨细胞分化后Runx2启动子区的甲基化水平。茜素红染色测定BMSCs向成骨细胞分化的程度。结果与诱导组相比,诱导+Sul组Runx2 mRNA和蛋白表达水平明显上升(P<0.05),与Histone H3结合的Runx2启动子区DNA含量增高(P<0.05),Runx2启动子区的甲基化水平降低(P<0.05),茜素红染色评分升高(P<0.05)。与诱导+Sul组相比,诱导+Sul+shRNA-TET1组细胞内与Histone H3结合的Runx2启动子区DNA含量降低(P<0.05),Runx2启动子区的甲基化水平升高(P<0.05),茜素红染色评分明显降低(P<0.05);而诱导+Sul+shRNA-TET2组、诱导+Sul+shRNA-TET3组及诱导+Sul+shRNA-Mock组则无明显变化(P>0.05)。结论Sul能够通过TET1促进Runx2启动子区DNA去甲基化,促进BMSCs向成骨细胞分化。
Objective To investigate the molecular mechanism of sulforaphane(Sul)promoting bone marrow stem cells(BMSCs)differentiating into osteoblasts.Methods BMSCs were divided into the control group(without any treatment),induction group(induction of osteogenic differentiation),and induction+Sul group(induction of osteogenic differentiation with the addition of 40µmol/L of Sul).The adenovirus-shRNA-Mock,-shRNA-TET1,-shRNA-TET2,and-shRNA-TET3 were transfected into BMSCs as the shRNA-Mock group,shRNA-TET1 group,shRNA-TET2 group,and shRNA-TET3 group.BMSCs were cultured in cell culture medium containing osteogenic differentiation induction medium and 40µmol/L of Sul,and then transfected with adenovirus-shRNA-TET1,-shRNA-TET2,-shRNA-TET3,and-shRNA-Mock as the induction+Sul+shRNA-TET1 group,induction+Sul+shRNA-TET2 group,induction+Sul+shRNA-TET3 group,and induction+Sul+shRNA-Mock group.The mRNA and protein expression levels of Runx2 after BMSCs differentiated into osteoblasts were determined by qPCR and Western blot.The DNA content of Runx2 promoter region bound to Histone H3 after BMSCs differentiated into osteoblasts was determined by chromatin immunocoprecipitation(ChIP).The methylation level of Runx2 promoter region of BMSCs differentiated into osteoblasts was determined by HpaⅡenzyme and MspⅠenzyme digestion combined with qPCR.The degree of BMSCs differentiated into osteoblasts was determined by alizarin red staining.Results Compared with the induction group,the mRNA and protein expression levels of Runx2 in the induction+Sul group were significantly increased(P<0.05);the content of DNA in the Runx2 promoter region bound to Histone H3 was increased(P<0.05),the methylation level of Runx2 promoter region was reduced(P<0.05),and the alizarin red staining score was elevated(P<0.05).Compared with the induction+Sul group,the content of DNA in the Runx2 promoter region bound to Histone H3 in the induction+Sul+shRNA-TET1 group was decreased(P<0.05),the methylation level of Runx2 promoter region was increased(P<0.05),and the
作者
张铮
韩佳雯
彭龙龙
聂涛
邹三明
张宇博
ZHANG Zheng;HAN Jia-wen;PENG Long-long;NIE Tao;ZOU San-ming;ZHANG Yu-bo(Department of BoneJoint and Sports Medicine,Xiaogan Hospital Affiliated to Wuhan University of Science and Technology,Xiaogan Hubei 432100,China;School of Medicine,Wuhan University of Science and Technology,Wuhan Hubei 430065,China)
出处
《局解手术学杂志》
2024年第1期24-29,共6页
Journal of Regional Anatomy and Operative Surgery
基金
湖北省卫生健康委员会科研项目(WJ2021M044)。
