地塞米松干预人骨髓间充质干细胞分化的表观遗传学修饰

maive To observe the effect of high-concentration dexamethasone on differentiation of human bone marrow mesenchymal stem cells through epigenetic modification. Methods Human bone marrow mesenchymal stem cells were cultured in a-MEM complete medium, expanded in vitro, and passed to the 4th generation. Microscopic electron micrographs were used to evaluate the cell growth state. The mRNA expressions of Runx2, ALP, OPG, RANKL, and Dnmtl after 3 d-intervention with different concentrations of dexamethasone ( 1 |±M or 10 jjlM ) were measured with real-time PCR. Protein expressions of Dnmtl, H3K4me3, and H3K27me3 were detected using Western blotting after 7 d treatment with 1 pimol/L of dexamethasone. Expressions of H3K4me3 and H3K27me3 antigen-antibody complex in the nucleus were detected with Immunofluorescence. After addition of DNA methylation inhibitor 5-Aza- 2 * -deoxycytidine intervention, mRNA expressions of Runx2, OPG, RANKL, Collal, and Dnmtl were observed. The effect of epigenetic related genes was analyzed. Results Real-time PCR result showed that early high concentration of (1 jimol/L or 10 p,mol/L) dexamethasone intervention promoted osteogenic differentiation of mesenchymal stem
mesenchymal stem cell by dexamethasone
WANG Peng1, WANG Haibin1,2* , XU Liangliang12, ZHANG Meng' , JIANG Shan' 1. Guangzhou University of Chinese Medicine, Guangzhou 510000, China 2. The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510000, China * Corresponding author: WANG Haibin, Email: hipknee@
骨髓间充质干细胞的分化效应。结论地塞米松对骨髓间充质干细胞分化的效应具有双向性，与干预时间有关，表观遗传相 关因子修饰也参与其作用的发生发展，这有助于从表观遗传学角度寻找治疗激素性骨质疏松的新方法。 关键词：地塞米松；表观遗传；骨髓间充质干细胞；骨质疏松
迁 Intervention of epigenetic mod ication in the differentiation of human bone
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地塞米松干预人骨髓间充质干细胞分化的表观遗传学 修饰
王鹏'王海彬I•“ 徐亮亮2 张濛'姜山' 1. 广州中医药大学，广东广州510000 2. 广州中医药大学第一附属医院，广东广州510000
中图分类号：R332 文献标识码：A 文章编号：1006-7108(2019) 11-1513-06 摘要：目的 观察高浓度地塞米松(dexamethasone, Dex)作用于人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, hBMSCs)的表观遗传修饰及对其分化作用的影响。方法 ①人骨髓间充质干细胞培养于a-MEM完全培养基中，体外扩 增，传至第4代，拍摄显微电镜图片评估细胞生长状态；②通过Real-time PCR检测经不同地塞米松浓度(1、10 pjnol/L)作用细 胞 3 d 后 Runx2、ALP、OPG、RANKL、Dnmtl( DNA methyltransferase 1)的 mRNA 表达；③Western-blot 检测经 1 jimol/L 浓度地塞 米松作用的细胞 7 d 后 Dnmtl、H3K4me3 ( trimethylation of lysine 4 on histone H3 )、H3K27me3 ( trimethylation of lysine27 on histone H3)蛋白表达；免疫荧光检测细胞核内H3K4me3、H3K27me3抗原-抗体复合物的表达；加予DNA甲基化抑制剂5-氮 杂-2** -脱氧胞昔(5-Aza-2'-deoxycytidine, 5-aza-dC)干预，观察 Runx2、0PG、RANKL、Collal、Dnmtl 的 mRNA 表达，分析表观 遗传相关基因在其中的作用。结果 ①Real-time PCR示高浓度(1 JO pimol/L)地塞米松对骨髓间充质干细胞干预早期有促 进其成骨分化的作用，Runx2、ALP、0PG、DNMTl基因表达增加；②Real-time PCR和Western-blot示中晚期1 pimol/L地塞米松 抑制间充质干细胞向成骨细胞分化；同时Dnmtl ,H3K27me3蛋白表达增加,H3K4me3表达下降；免疫荧光检测示细胞核内 H3K4me3、H3K27me3抗原-抗体复合物表达情况与Western-blot结果类似；③DNA甲基化抑制剂5-aza-dC可影响地塞米松对
中国骨质疏松杂志 2019 年 11 月第 25 卷第 11 期 Chin J Osteoporos, November 2019, Vol 25 , No. 11 doi ： 10. 3969/j.issn. 1006-7108. 2019. 11.001