丙戊酸促进大鼠骨髓间充质干细胞的成骨分化  

Valproic acid promotes osteogenic differentiation of rat bone marrow mesenchymal stem cells

作　　者：凌徐玮 孙杰 刘畅[1,2] 王怡[1,2] 施勤[1,2] 杨惠林 Ling Xuwei;Sun Jie;Liu Chang;Wang Yi;Shi Qin;Yang Huilin(Department of Orthopedics,First Affiliated Hospital of Soochow University,Suzhou Medical College of Soochow University,Suzhou 215006,Jiangsu Province,China;Institute of Orthopedics,Soochow University,Suzhou 215006,Jiangsu Province,China)

机构地区：[1]苏州大学附属第一医院骨科,苏州大学苏州医学院,江苏省苏州市215006 [2]苏州大学骨科研究所,江苏省苏州市215006

出　　处：《中国组织工程研究》2023年第15期2304-2310,共7页Chinese Journal of Tissue Engineering Research

基　　金：卫生厅“科教兴卫”项目-江苏省骨科临床医学中心,项目负责人:杨惠林。

摘　　要：背景:丙戊酸作为一种组蛋白去乙酰化酶抑制剂,具有促进细胞成骨分化的能力,但相关机制尚不明确。目的:探讨丙戊酸对大鼠骨髓间充质干细胞成骨分化的作用及机制。方法:从SD大鼠中分离提取骨髓间充质干细胞,流式细胞仪进行鉴定;CCK-8检测不同浓度丙戊酸对大鼠骨髓间充质干细胞增殖的影响,以确定丙戊酸的使用浓度;然后加入丙戊酸和成骨培养基对大鼠骨髓间充质干细胞成骨诱导分化,在成骨诱导培养第7天进行碱性磷酸酶染色以及第21天进行茜素红染色;成骨诱导培养第3,7天进行qRT-PCR检测成骨相关基因及EZH2的表达;成骨诱导培养第5天进行Western blot检测Runt相关转录因子2、EZH2和H3K27me3相关蛋白的表达。结果与结论:①CCK-8细胞增殖实验显示,随着丙戊酸浓度的增加,骨髓间充质干细胞增殖受到显著抑制;②碱性磷酸酶染色及茜素红染色结果显示,丙戊酸能提高细胞碱性磷酸酶的表达,增强骨矿化能力;③qRT-PCR结果显示,丙戊酸可提高成骨相关基因的表达,降低EZH2基因的表达;④Western blot结果显示,丙戊酸使Runt相关转录因子2蛋白表达增强,EZH2和H3K27me3蛋白表达下降;⑤结果表明,丙戊酸能够通过下调EZH2的表达促进大鼠骨髓间充质干细胞的成骨分化。BACKGROUND:Valproic acid,as a histone deacetylase inhibitor,has the ability to promote osteogenic differentiation of cells,but the mechanism is still unclear.OBJECTIVE:To investigate the effect and mechanism of valproic acid on osteogenic differentiation of rat bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and identified by flow cytometry.CCK-8 assay was used to detect the effects of different concentrations of valproic acid on the proliferation of rat bone marrow mesenchymal stem cells,and determine the used concentration of valproic acid.After adding valproic acid,rat bone marrow mesenchymal stem cells were induced to osteogenic differentiation.Alkaline phosphatase staining was performed on day 7 of osteogenic induction culture,and alizarin red staining was performed on day 21 of osteogenic induction culture.Quantitative real-time polymerase chain reaction was used to detect the expression of osteogenesis-related genes and EZH2 on days 3 and 7 of osteogenic induction culture.The protein expression levels of Runt-related transcription factor 2,EZH2 and H3K27me3 were detected by western blot assay on day 5 of osteogenic induction culture.RESULTS AND CONCLUSION:(1)CCK-8 cell proliferation assay showed that bone marrow mesenchymal stem cell proliferation was significantly inhibited by increasing the concentration of valproic acid.(2)The results of alkaline phosphatase staining and alizarin red staining showed that valproic acid could increase the expression of alkaline phosphatase and enhance the ability of bone mineralization.(3)qRT-PCR results showed that valproic acid could increase the expression of osteogenesis-related genes and decrease the expression of EZH2 gene.(4)Western blot assay results showed that valproic acid could increase the expression of Runt-related transcription factor 2 protein and decrease the expression of EZH2 and H3K27me3 protein.(5)It is concluded that valproic acid can promote osteogenic differentiation of rat bone m

关 键 词：丙戊酸 EZH2 H3K27me3 骨髓间充质干细胞 成骨分化 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学] R318