MeCP2在增龄性EPCs功能障碍中的作用及机制

Methyl-CpG-binding protein 2 (MeCP2), a methylation dependent transcriptional repressor, is widely expressed in various organic tissues and cell types. MeCP2 is deregulated in many pathological conditions. To date, however, a role for MeCP2 that plays in the regulation of endothelial progenitor cells (EPCs) function has been completely unknown. Previous study from our group have shown that the expression level of MeCP2 is markedly increased, whereas the level of SIRT1, which has positive effects on EPCs function is decreased in senescent EPCs. Therefore, we postulate that MeCP2 may impair EPCs function via transcriptional repression of SIRT1. To test this hypothesis, experimental techniques such as Chromatin Immunoprecipitation, Real-time quantitative PCR, adenoviral mediated gene overexpression and RNA interference are to be used. By virtue of using human endothelial progenitor cells, model of aging rats and ApoE-/- mice atherosclerotic animal model, we aimed to determine the role for MeCP2 in dysfunction of senescent EPCs, to confirm MeCP2 induced EPCs dysfunction via repression of SIRT expression, and to further explore the mechanism(s) of SIRT1 regulation by MeCP2, from molecular, cellular to whole animal level. The present study may help lay a foundation for revealing the mechanisms of ageing EPCs dysfunction from the new viewpoint of MeCP2 and may also provide a new concept for the prevention and treatment of arterial atherosclerotic diseases.

MeCP2是广泛表达于机体各组织细胞的一种转录抑制因子，在许多病理情况下存在MeCP2的表达失调。迄今MeCP2对EPCs的调控作用尚不清楚。我们前期研究结果表明：SIRT1对EPCs功能具有正向调控作用，衰老EPCs中MeCP2的表达水平升高而SIRT1水平下降。因此我们提出MeCP2可能通过抑制SIRT1表达影响EPCs功能的假设。为验证这一假设，我们将通过人EPCs、大鼠衰老模型和小鼠动脉粥样硬化模型，采用染色质免疫共沉淀、定量PCR、RNA干扰、基因过表达、Westernblot等手段，从分子、细胞、动物整体水平多方面明确MeCP2在衰老EPCs功能障碍中的作用，证实MeCP2通过抑制SIRT1表达影响EPCs功能，并进一步探讨MeCP2对SIRT1的调控机制。本项目将从MeCP2这个新视点为揭示增龄性EPCs功能障碍的机制奠定基础，为动脉粥样硬化性疾病的防治提供新的思路。

EPCs衰老是血管衰老性疾病形成、发展过程中的初始变化和主要环节，然而衰老EPCs功能障碍的机制尚不清楚。本研究首先提取自然衰老大鼠的外周血EPCs和健康产妇的脐血EPCs，并采用持续传代和H2O2建立复制性衰老和应激性早衰模型，通过CCK-8、细胞周期实验、流式凋亡实验、Transwell小室、划痕和基质胶小管实验发现随EPCs衰老，细胞活性降低、增殖减弱、凋亡增加、迁移及血管形成能力受损，同时伴有MeCP2表达升高和SIRT1表达下降。然后构建过表达/沉默MeCP2和SIRT1的腺病毒并转染EPCs后发现：SIRT1正向调控EPCs功能，而过表达MeCP2降低细胞功能和SIRT1表达，沉默MeCP2提高细胞功能和SIRT1表达，共转染MeCP2和SIRT1则可逆转，表明EPCs功能障碍与MeCP2表达升高并抑制SIRT1有关。最后探讨MeCP2对SIRT1的调控机制，通过亚硫酸氢钠甲基化测序和染色质免疫共沉淀实验发现MeCP2靶向促进SIRT1启动子区的DNA和组蛋白H3K9甲基化，从而抑制SIRT1转录。本课题为衰老EPCs功能障碍的机制提供了一个新的研究靶点，对防治血管衰老性疾病具有重要意义。