【作者】 李娜；

【导师】 齐颖新；

【作者基本信息】 上海交通大学 ， 生物学， 2018， 硕士

【摘要】 内皮祖细胞(Endothelial progenitor cells,EPCs)又称为成血管细胞,是具有自我更新能力并可分化为成熟内皮细胞(endothelial cells,ECs)的多能干细胞,在内膜功能的修复和血管稳态维持过程中发挥关键作用。血管内膜发生损伤后,EPCs被动员并迁移、粘附至损伤部位,粘附在血管壁上的EPCs会受到血流脉动产生的周期性张应变(cyclic stretch)的作用,对EPC分化产生重要的影响。目前认为MicroRNAs(miRNAs)是多种胞内活动的关键因素,然而其在周期性张应变诱导的EPC分化过程中的作用尚不明确。本文对骨髓来源的EPCs施加幅度为5%,频率为1.25 Hz生理水平的周期性张应变,加载时间24 h。应用miRNAs芯片检测周期性张应变调控下的miRNAs的表达情况,挑选出表达量发生显著变化的两种miRNAs:microRNA-129-1-3p(miR-129-1-3p)和microRNA-214-3p(miR-214-3p),深入探讨了它们在周期性张应变诱导的EPC分化过程中的作用。结果显示,周期性张应变显著抑制miR-129-1-3p表达,同时诱导EPCs向EC相分化,并抑制其向血管平滑肌(vascular smooth muscle cells,VSMCs)相分化。靶基因预测网站TargetScan(http://www.targetscan.org)和miRanda(http://www.microrna.org)依据结合位点预测miR-129-1-3p的下游调控靶基因Runt-related transcription factor 2(Runx2)。周期性张应变促进了miR-129-1-3p的潜在靶基因Runx2的表达。双荧光素酶报告基因实验证实,miR-129-1-3p直接靶向Runx2。之后,使用特异性的siRNA片段干扰Runx2,结果表明Runx2水平下降会抑制EPCs向EC相分化,并减少EPCs在体外基质胶微管的形成。此外,周期性张应变促进EPCs分泌血管内皮生长因子(vascular endothelial growth factor,VEGF),同时miR-129-1-3p表达增加或Runx2水平下降会导致VEGF分泌量减少。此外,周期性张应变显著抑制EPCs内miR-214-3p的表达,抑制EPCs中miR-214-3p表达则会促进细胞增殖并抑制EPCs向VSMC相分化。我们的研究揭示,生理性的周期性张应变会改变EPCs中miRNAs的表达,并通过调控其靶基因最终影响EPCs增殖、分化以及血管生成的能力。综上所述,寻找关键性的miRs及其下游信号通路,能够为血管损伤修复提供了新的治疗依据。更多还原

【Abstract】 Endothelial progenitor cells(EPCs)are vital to the recovery of endothelial function and maintenance of vascular homeostasis through mobilizing to the sites of vessel injury and differentiating into mature endothelial cells(ECs).Once the tissue is damaged,bone marrow(BM)derived EPCs are recruited and migrate to the injury sites.EPCs are exposed to cyclic stretch imposed by blood flow,which plays important roles in modulating differentiation of EPCs.MicroRNAs(miRNAs)have emerged as key regulators of several cellular processes.However,the role of miRNAs in EPC differentiation induced by cyclic stretch is still unclear.BM-derived EPCs were exposed to cyclic stretch with the magnitude of 5%,which mimics the physiological stretch,at a constant frequency of 1.25 Hz for 24 hr.miRNAs array was performed to identify the expression profiling of miRNAs regulated by mechanical stretch.Here we set out to investigate the effects of microRNA-129-1-3p(miR-129-1-3p)and microRNA-214-3p(miR-214-3p).The results indicated that cyclic stretch significantly decreased miR-129-1-3p expression and induced EPC differentiation toward ECs,depressed EPC differentiation toward vascular smooth muscle cells(VSMCs).Runt-related transcription factor 2(Runx2),a putative target gene of miR-129-1-3p,was predicted by TargetScan(http://www.targetscan.org)and miRanda(http://www.microrna.org).Runx2,was increased by cyclic stretch.Dual luciferase reporter assay validated Runx2 as a direct target of miR-129-1-3p.Furthermore,siRNA-mediated knockdown of Runx2 inhibited EPC differentiation into ECs and attenuated tube formation of EPCs by modulating vascular endothelial growth factor(VEGF)secretion from EPCs in vitro.Furthermore,cyclic stretch depresses the expression of miR-214-3p significantly,enhance EPC proliferation and inhibits EPC differentiation towards VSMCs.Our findings demonstrated that physiological cyclic stretch regulates the expression of miRs and their target genes,which in turn promote endothelial differentiation of EPCs,proliferation and angiogenic ability.Thus,targeting key miRs may be a potential therapeutic strategy for treating vessel injury.更多还原