三维和二维培养的脐带间充质干细胞DNA甲基化水平比较

doi:10.6040/j.issn.1671-7554.0.2019.813

摘要/Abstract

摘要： 目的探讨三维培养对脐带间充质干细胞(UC-MSCs)全基因组DNA甲基化状态的影响。方法通过贴壁培养法获得UC-MSCs,采用流式细胞术鉴定其细胞表型。采用聚氟乙烯巢状片进行三维培养,吖啶橙荧光观察UC-MSCs在支架上生长情况。采用甲基化DNA免疫共沉淀测序(MeDIP-Seq)技术检测细胞全基因组DNA甲基化情况。结果成功分离培养UC-MSCs并证明其表型符合国际标准,细胞黏附在聚氟乙烯巢状片三维培养体系中且生长良好。MeDIP-Seq结果显示,三维培养后UC-MSCs全基因组DNA甲基化水平较二维培养整体上调。基因本体(GO)分析显示,与二维培养的细胞相比,三维培养的UC-MSCs中上调的差异甲基化基因多参与蛋白质的代谢、膜相关细胞器的组成、酶活性调节等;而下调的差异甲基化基因主要参与囊泡运输和cGMP代谢过程。通路分析显示,三维培养的UC-MSCs中上调的差异甲基化基因多参与RNA转运、神经配体受体活性、Notch信号通路等;下调的差异甲基化基因多参与磷酸肌醇代谢通路和囊泡分泌相关的通路。结论三维培养改变了UC-MSCs的DNA甲基化谱,所涉及的甲基化上调基因多参与细胞的迁移和增殖功能。三维培养降低了UC-MSCs的免疫调控和分泌囊泡相关基因的甲基化水平。

关键词: 脐带间充质干细胞, 三维培养, DNA甲基化, 甲基化DNA免疫共沉淀测序

Abstract: Objective To explore the effects of three-dimensional(3D)culture on the whole genome DNA methylation profile of umbilical cord mesenchymal stem cells(UC-MSCs). Methods UC-MSCs were isolated from umbilical cords using tissue adherent culture. Their phenotypes were identified with flow cytometry. Then UC-MSCs were cultured in 3D polyvinyl fluoride nested scaffolds and 2D conventional culture flask, respectively. Acridine orange was used to observe UC-MSCs on the scaffold. Methylated DNA immunoprecipitation(MeDIP-Seq)method was used to detect the whole genome DNA methylation of UC-MSCs. Results UC-MSCs were successfully isolated from umbilical cords and able to grow on the 3D scaffold. The genomic DNA methylation level of UC-MSCs increased after 3D culture. Gene ontology analysis showed that the up-regulated differential methylation genes were involved in protein metabolism, membrane-related organelle composition, and enzyme activity regulation. Down-regulated differential methylation genes were involved in vesicle trafficking and cGMP metabolic processes regulating. According to pathway analyses, differential methylation genes were involved in RNA transport, neuroactive ligand-receptor interaction, cellular communication, and 山东大学学报 (医学版)57卷11期 -黄金献,等.三维和二维培养的脐带间充质干细胞DNA甲基化水平比较 \=-vesicle secretion-related pathways. Conclusion 3D culture changes the DNA methylation profile of UC-MSCs. The up-regulated methylation levels will alter the function of cell migration and proliferation. 3D culture reduces the methylation level of immune regulation and secretion of vesicle-related genes in UC-MSCs.

Key words: Mesenchymal stromal cells, Three-dimensional culture, DNA methylation, methylated DNA immunoprecipitation sequencing

中图分类号:

R574

黄金献,李栋,李聪,时庆,鞠秀丽. 三维和二维培养的脐带间充质干细胞DNA甲基化水平比较[J]. 山东大学学报 (医学版), 2019, 57(11): 1-8.

HUANG Jinxian, LI Dong, LI Cong, SHI Qing, JU Xiuli. Comparison of DNA methylation levels between three-dimensional and two-dimensional cultured umbilical cord mesenchymal stem cells[J]. Journal of Shandong University (Health Sciences), 2019, 57(11): 1-8.

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