基质细胞衍生因子-1α对心脏祖细胞标志物 c-kit 的表观遗传调控。
Epigenetic regulation of cardiac progenitor cells marker c-kit by stromal cell derived factor-1α.
机构信息
Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu, China.
出版信息
PLoS One. 2013 Jul 24;8(7):e69134. doi: 10.1371/journal.pone.0069134. Print 2013.
BACKGROUND
Cardiac progenitor cells (CPCs) have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+)CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF), are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α) could enhance the expression of c-kit. However, the mechanism is unknown.
METHODS AND RESULTS
CPCs were isolated from adult mouse hearts, c-kit(+) and c-kit(-) CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+)CPCs, made c-kit(-)CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+) and c-kit(-) CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT) mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+) and c-kit(-) CPCs.
CONCLUSIONS
SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+)CPCs and make c-kit(-)CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT activity, as well as the subsequent demethylation of the c-kit gene.
背景
心脏祖细胞（CPCs）已被证明适合心肌梗死后的干细胞治疗，尤其是 c-kit（+）CPCs。CPCs 标志物 c-kit 及其配体干细胞因子（SCF）构成 c-kit/SCF 轴，与增殖和分化功能相关。在我们之前的研究中，我们发现基质细胞衍生因子-1α（SDF-1α）可以增强 c-kit 的表达。然而，其机制尚不清楚。
方法和结果
从成年小鼠心脏中分离 CPCs，用磁珠分离 c-kit（+）和 c-kit（-）CPCs。用 SDF-1α 和 CXCR4 选择性拮抗剂 AMD3100 培养细胞，通过 qPCR 和 Western blot 测量 c-kit 的表达。结果表明，SDF-1α 可以增强 c-kit（+）CPCs 的 c-kit 表达，使 c-kit（-）CPCs 表达 c-kit，AMD3100 可以抑制 SDF-1α 的功能。在 SDF-1α 和 AMD3100 干预后，通过 CCK-8 和 Transwell 测定测量 CPCs 的增殖和迁移。结果表明，SDF-1α 可以增强 c-kit（+）和 c-kit（-）CPCs 的增殖和迁移，AMD3100 可以抑制这些功能。用 SDF-1α 培养 CPCs 后，通过 qPCR 测量 DNA 甲基转移酶（DNMT）mRNA，使用 DNMT 活性测定试剂盒测量 DNMT 活性，并使用 Sequenom 的 MassARRAY 平台分析 DNA 甲基化。结果表明，SDF-1α 刺激抑制了区域 DNA 甲基化维持所必需的 DNMT1 和 DNMT3β 的表达。SDF-1α 还抑制了全局 DNMT 活性。最后，SDF-1α 处理导致 c-kit（+）和 c-kit（-）CPCs 中的显著去甲基化。
结论
SDF-1α 与 CXCR4 结合可上调 c-kit（+）CPCs 的 c-kit 表达，并使 c-kit（-）CPCs 表达 c-kit，从而通过抑制 DNMT1 和 DNMT3β 的表达和全局 DNMT 活性以及随后的 c-kit 基因去甲基化，改善 CPCs 的增殖和迁移能力。
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