SIRT1与microRNA-34a在切应力诱导内皮祖细胞分化中的作用及其机制
发布时间：2018-10-22 09:35
【摘要】：内皮祖细胞（endothelial progenitor cells, EPCs）分化在血管内膜损伤修复和血管稳态维持过程中均具有重要作用。血流产生的切应力（shear stress）是调控EPC分化的关键因素之一，但其相关的力学生物学（mechanobiology）机制尚需进一步阐明。 本文应用平行平板流动腔系统对人脐血来源的EPCs施加15dyn/cm2生理水平的层流切应力，作用时间24h，首先证明了切应力促进EPCs向成熟的内皮细胞（endothelial cells, ECs）分化，且抑制其向平滑肌细胞（smooth muscular cells, SMCs）分化。为了进一步探讨切应力条件下EPC分化的相关力学生物学机制，我们开展了以下2部分研究： 在第一部分，首先研究了SIRT1在切应力调控EPC分化中的作用机制。结果显示，切应力明显地诱导了EPCs的Akt磷酸化活性、SIRT1表达升高、组蛋白H3在赖氨酸9位点的乙酰化（ac-H3K9）降低，且三者依次在6、12和24h达到峰值。转染SIRT1特异性siRNA下调了EPCs的EC标志分子KDR、VE-cadherin、vWF和CD31的表达，促进SMC标志分子α-SMA和sm22α的表达，同时增强了组蛋白H3的乙酰化水平；而SIRT1激活剂白藜芦醇（resveratrol）对EPC分化标志表达和组蛋白H3乙酰化作用的结果与之相反。用PI3K的抑制剂wortmannin孵育EPCs，抑制其EC标志分子，却促进了其SMC标志分子的表达，，并下调了SIRT1表达，增强了组蛋白H3的乙酰化。此外，体外基质胶微管形成（tube formation）实验表明，SIRT1促进EPCs微管状结构的形成与连接。上述结果提示，PI3K/Akt-SIRT1-ac-H3K9信号通路可能参与了切应力诱导的EPCs向ECs的分化。 在第二部分，我们探讨了microRNA-34a（miR-34a）在切应力调控EPC分化中的可能分子机制。结果显示，切应力时间依赖地诱导EPCs的miR-34a表达，并在12h达到峰值；应用3种靶基因预测数据库预测提示，FOXJ2可能作为miR-34a的潜在靶基因参与细胞功能调控。双荧光报告基因实验证明，FOXJ2是miR-34a的直接靶基因。然后，分别应用miR-34a mimics和inhibitor刺激EPCs，进一步验证了miR-34a对FOXJ2的负向调节；并且切应力对FOXJ2的表达具有抑制作用。miR-34a正向调控EPCs的EC标志分子KDR、VE-cadherin、vWF和CD31的表达而负向调控SMC标志分子α-SMA、sm22α、calponin和smmhc的表达。之后，进一步过表达或干扰FOXJ2，结果显示，FOXJ2对EPC分化的作用与miR-34a的作用相反。此外，干扰FOXJ2促进了EPCs在体外基质胶的微管形成。上述结果提示，miR-34a可能通过负向调控其靶基因FOXJ2，参与了切应力诱导的EPCs向ECs分化。 综上所述，生理水平的层流切应力促进了EPCs向ECs分化而抑制其向SMCs分化；PI3K/Akt-SIRT1-ac-H3K9和miR-34a-FOXJ2可能是其中重要的信号通路。这些研究结果对阐明EPCs响应力学刺激的信号转导机制有重要意义，也为血管损伤修复和缺血性疾病的治疗提供了新的力学生物学思路。
[Abstract]:Endothelial progenitor cell (endothelial progenitor cells, EPCs) differentiation plays an important role in the repair of vascular intima injury and the maintenance of vascular homeostasis. The shear stress (shear stress) produced by blood flow is one of the key factors in regulating the differentiation of EPC, but the related mechanism of (mechanobiology) in mechanical biology needs to be further elucidated. In this paper, a parallel plate flow chamber system was used to apply laminar shear stress at the physiological level of 15dyn/cm2 to EPCs derived from human umbilical cord blood for 24 h. It was proved that shear stress could promote the differentiation of EPCs into mature endothelial cell (endothelial cells, ECs). And inhibit its differentiation to smooth muscle cell (smooth muscular cells, SMCs). In order to further study the mechanics and biological mechanism of EPC differentiation under shear stress, we studied the following two parts: in the first part, we studied the mechanism of SIRT1 in regulating the differentiation of EPC under shear stress. The results showed that shear stress induced the Akt phosphorylation activity of EPCs, the expression of SIRT1 increased, and the acetylation (ac-H3K9) of histone H3 decreased at lysine 9 site, which reached the peak at 612 and 24 h, respectively. SIRT1 specific siRNA down-regulated the expression of KDR,VE-cadherin,vWF and CD31, promoted the expression of SMC marker 伪 -SMA and sm22 伪, and enhanced the acetylation level of histone H3. The effect of resveratrol (resveratrol), a SIRT1 activator, on the expression of EPC differentiation markers and histone H 3 acetylation was reversed. EPCs, was incubated with wortmannin, an inhibitor of PI3K, to inhibit the expression of EC markers, but to promote the expression of SMC markers, down-regulate the expression of SIRT1 and enhance the acetylation of histone H3. In addition, SIRT1 promoted the formation and connection of EPCs microtubules by (tube formation). These results suggest that the PI3K/Akt-SIRT1-ac-H3K9 signaling pathway may be involved in the differentiation of EPCs into ECs induced by shear stress. In the second part, we investigate the possible molecular mechanism of microRNA-34a (miR-34a) in regulating the differentiation of EPC by shear stress. The results showed that the miR-34a expression of EPCs was induced in a time-dependent manner and reached its peak at 12 h. The results of three target gene prediction databases suggested that FOXJ2 might be involved in the regulation of cell function as a potential target gene of miR-34a. Double fluorescence reporter gene experiment proved that FOXJ2 is a direct target gene of miR-34a. Then, miR-34a mimics and inhibitor were used to stimulate EPCs, to further verify the negative regulation of FOXJ2 by miR-34a. Shear stress inhibited the expression of FOXJ2. MiR-34a positively regulated the expression of EC marker KDR,VE-cadherin,vWF and CD31 of EPCs, while negatively regulated the expression of SMC marker 伪 -SMA,sm22 伪, calponin and smmhc. After that, further overexpression or interference with FOXJ2, showed that the effect of FOXJ2 on EPC differentiation was contrary to that of miR-34a. In addition, interfering with FOXJ2 promoted the formation of microtubules of EPCs in vitro. These results suggest that miR-34a may participate in the shear stress induced differentiation of EPCs into ECs through negative regulation of its target gene FOXJ2,. In conclusion, laminar shear stress at physiological level promotes the differentiation of EPCs into ECs and inhibits its differentiation to SMCs, and PI3K/Akt-SIRT1-ac-H3K9 and miR-34a-FOXJ2 may be important signaling pathways. These results have important significance in elucidating the signal transduction mechanism of EPCs in response to mechanical stimulation, and also provide a new biomechanical biological idea for vascular injury repair and the treatment of ischemic diseases.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.01
本文编号：2286792
[Abstract]:Endothelial progenitor cell (endothelial progenitor cells, EPCs) differentiation plays an important role in the repair of vascular intima injury and the maintenance of vascular homeostasis. The shear stress (shear stress) produced by blood flow is one of the key factors in regulating the differentiation of EPC, but the related mechanism of (mechanobiology) in mechanical biology needs to be further elucidated. In this paper, a parallel plate flow chamber system was used to apply laminar shear stress at the physiological level of 15dyn/cm2 to EPCs derived from human umbilical cord blood for 24 h. It was proved that shear stress could promote the differentiation of EPCs into mature endothelial cell (endothelial cells, ECs). And inhibit its differentiation to smooth muscle cell (smooth muscular cells, SMCs). In order to further study the mechanics and biological mechanism of EPC differentiation under shear stress, we studied the following two parts: in the first part, we studied the mechanism of SIRT1 in regulating the differentiation of EPC under shear stress. The results showed that shear stress induced the Akt phosphorylation activity of EPCs, the expression of SIRT1 increased, and the acetylation (ac-H3K9) of histone H3 decreased at lysine 9 site, which reached the peak at 612 and 24 h, respectively. SIRT1 specific siRNA down-regulated the expression of KDR,VE-cadherin,vWF and CD31, promoted the expression of SMC marker 伪 -SMA and sm22 伪, and enhanced the acetylation level of histone H3. The effect of resveratrol (resveratrol), a SIRT1 activator, on the expression of EPC differentiation markers and histone H 3 acetylation was reversed. EPCs, was incubated with wortmannin, an inhibitor of PI3K, to inhibit the expression of EC markers, but to promote the expression of SMC markers, down-regulate the expression of SIRT1 and enhance the acetylation of histone H3. In addition, SIRT1 promoted the formation and connection of EPCs microtubules by (tube formation). These results suggest that the PI3K/Akt-SIRT1-ac-H3K9 signaling pathway may be involved in the differentiation of EPCs into ECs induced by shear stress. In the second part, we investigate the possible molecular mechanism of microRNA-34a (miR-34a) in regulating the differentiation of EPC by shear stress. The results showed that the miR-34a expression of EPCs was induced in a time-dependent manner and reached its peak at 12 h. The results of three target gene prediction databases suggested that FOXJ2 might be involved in the regulation of cell function as a potential target gene of miR-34a. Double fluorescence reporter gene experiment proved that FOXJ2 is a direct target gene of miR-34a. Then, miR-34a mimics and inhibitor were used to stimulate EPCs, to further verify the negative regulation of FOXJ2 by miR-34a. Shear stress inhibited the expression of FOXJ2. MiR-34a positively regulated the expression of EC marker KDR,VE-cadherin,vWF and CD31 of EPCs, while negatively regulated the expression of SMC marker 伪 -SMA,sm22 伪, calponin and smmhc. After that, further overexpression or interference with FOXJ2, showed that the effect of FOXJ2 on EPC differentiation was contrary to that of miR-34a. In addition, interfering with FOXJ2 promoted the formation of microtubules of EPCs in vitro. These results suggest that miR-34a may participate in the shear stress induced differentiation of EPCs into ECs through negative regulation of its target gene FOXJ2,. In conclusion, laminar shear stress at physiological level promotes the differentiation of EPCs into ECs and inhibits its differentiation to SMCs, and PI3K/Akt-SIRT1-ac-H3K9 and miR-34a-FOXJ2 may be important signaling pathways. These results have important significance in elucidating the signal transduction mechanism of EPCs in response to mechanical stimulation, and also provide a new biomechanical biological idea for vascular injury repair and the treatment of ischemic diseases.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.01
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