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  

Intervention of epigenetic modification in the differentiation of human bone marrow mesenchymal stem cell by dexamethasone

作　　者：王鹏[1] 王海彬[1,2] 徐亮亮 张濛 姜山 WANG Peng;WANG Haibin;XU Liangliang;ZHANG Meng;JIANG Shan(Guangzhou University of Chinese Medicine,Guangzhou 510000,China;The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine,Guangzhou 510000,China)

机构地区：[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

出　　处：《中国骨质疏松杂志》2019年第11期1513-1517,1565,共6页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助项目(81774339)

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ɑ-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 μmol/L)作用细胞3 d后Runx2、ALP、OPG、RANKL、Dnmt1(DNA methyltransferase 1)的mRNA表达;③Western-blot检测经1 μmol/L浓度地塞米松作用的细胞7 d后Dnmt1、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’-脱氧胞苷(5-Aza-2'-deoxycytidine, 5-aza-dC)干预,观察Runx2、OPG、RANKL、Col1a1、Dnmt1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、10 μmol/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、OPG、DNMT1基因表达增加;②Real-time PCR和Western-blot示中晚期1 μmol/L地塞米松抑制间充质干细胞向成骨细胞分化;同时Dnmt1、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。Objective 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 ɑ-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 Dnmt1 after 3 d-intervention with different concentrations of dexamethasone (1 μM or 10 μM) were measured with real-time PCR. Protein expressions of Dnmt1, H3K4me3, and H3K27me3 were detected using Western blotting after 7 d treatment with 1 μmol/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, Col1a1, and Dnmt1 were observed. The effect of epigenetic related genes was analyzed. Results Real-time PCR result showed that early high concentration of (1 μmol/L or 10 μmol/L) dexamethasone intervention promoted osteogenic differentiation of mesenchymal stem cells, and the expressions of Runx2, ALP, OPG and DNMT1 genes increased. Real-time PCR and Western blotting result showed that middle and late intervention with 1 μmol/L of dexamethasone inhibited the osteogenic differentiation of mesenchymal stem cells, while the expressions of Dnmt1 and H3K27me3 protein increased, but the expression of H3K4me3 decreased. Immunofluorescence result showed that the expression of H3K4me3 and H3K27me3 antigen-antibody complexes in the nucleus was consistent to that of Western blotting result . DNA methylation inhibitor 5-aza-dC affected the differentiation of bone marrow mesenchymal stem cells by dexamethasone. Conclusion These result indicate that the effect of dexamethasone on the differentiation of mesenchymal stem cells is bidirectional, which is related to the intervention time. Epigenetic re

关 键 词：地塞米松 表观遗传 骨髓间充质干细胞 骨质疏松 

分 类 号：R332[医药卫生—人体生理学]