ABCB1基因过甲基化修饰在激素性股骨头坏死中的作用  

Role of ABCB1 gene hypermethylation of mesenchymal stem cells in steroid-associated osteonecrosis

作　　者：张山锋 马中希 叶志伟 刘珍星 杨钟华 ZHANG Shan-feng;MA Zhong-xi;FE Zhi-wei;LIU Zhen-xing;YANG Zhong-hua(Department of Orthopedics,Wuhan Fourth Hospital,Puai Hospital.Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430034,Chin)

机构地区：[1]武汉市第四医院华中科技大学同济医学院附属普爱医院骨科,武汉430034

出　　处：《中国矫形外科杂志》2018年第13期1221-1226,共6页Orthopedic Journal of China

基　　金：武汉市卫计委资助项目(编号:WX16C28)

摘　　要：[目的]探讨ABCB1基因过甲基化修饰对激素性股骨头坏死(ONFH)骨髓间充质干细胞(MSCs)功能失调的影响。[方法]选取18例激素性股骨头坏死患者和18例的股骨颈骨折患者,无菌条件下抽取患者股骨骨髓,密度梯度离心法体外分离培养MSCs,取第三代细胞实验。实验分三组:ONFH组(骨坏死患者的MSCs)、5′-氮杂胞苷组(ONFH组的细胞经5′-氮杂胞苷干预72 h)、正常对照组(股骨颈骨折患者的MSCs)。分别检测3组细胞内活性氧(ROS)、线粒体膜电位(MMP)。提取全基因组DNA,亚硫酸氢盐测序(BSP)检测ABCB1基因启动子区的甲基化情况。[结果]与正常对照组相比,ONFH组MSCs增殖能力下降、ROS上升、MMP降低,差异有统计学意义(P<0.05)。15μmol/L 5′-氮杂胞苷干预培养后能明显促进ONFH的MSCs增殖能力,降低ROS,提高细胞内的MMP,并且接近正常对照组水平,5′-氮杂胞苷组与ONFH组差异有统计学意义(P<0.05)。亚硫酸氢盐测序结果显示:激素性股骨头坏死患者MSCs的ABCB1基因启动子甲基化程度明显高于对照组(P<0.05),15μmol/L 5′-氮杂胞苷处理后甲基化程度接近正常对照组水平(P<0.05)。[结论]激素性股骨头坏死骨髓间充质干细胞中存在ABCB1基因过甲基化修饰,并导致其功能失调。[Objective] To investigate the role of ABCB1 gene hypermethylation of mesenchymal stem cells in steroid-associated osteonecrosis. [Methods] Bone marrow was harvested from the proximal femur of 18 patients with glucocorticoid（GC）-associated osteonecrosis of the femoral head（ONFH） and 18 patients with new femoral neck fractures. The mesenchymal stem cells（MSCs） were isolated by ficoll density-gradient centrifugation. The third generation cells digested and inoculated in 6-well plates were used for this study, including the ONFH group（the cells from ONFH）, the 5＇-Aza-d C treated ONFH group（the cells from ONFH previously treated for 72 h with 5＇-Aza-2-deoxycytidine, a DNA methyltransferase inhibitor） and the normal control group（the cells from fresh neck-fractured femoral head）. The viability, intracellular reactive oxygen species（ROS） level and mitochondrial membrane potential（MMP） of the cells were assessed in the 3 groups.In addition,genome DNA was extracted, and then methylation at Cp G islands of ABCB1 promoter was investigated by bisulfite sequencing.[Results]The MSCs from the ONFH groups showed significantly reduced proliferation capacity, elevated ROS level and depressed MMP compared with the normal control groups（P〈0.05）. By contrast,the MSCs in the 5＇-Aza-d C treated ONFH group were of significantly higher proliferation, lower ROS level and higher MMP than those in the ONFH group（P〈0.05）. In term of bisulfite sequencing, the ONFH group had significantly higher level of ABCB1 gene promoter methylation than the normal control group（P〈0.05）, which rapidly decreased to the normal level as treated with 5＇-Aza-d C（P〈0.05）. [Conclusion] The results of this study suggest that the hypermethylation of ABCB1 gene could be related to the development of GC-induced ONFH.

关 键 词：ABCB1基因 甲基化 激素性股骨头坏死 骨髓间充质干细胞 

分 类 号：R318[医药卫生—生物医学工程]