SIRT1與microRNA-34a在切應(yīng)力誘導(dǎo)內(nèi)皮祖細(xì)胞分化中的作用及其機(jī)制
發(fā)布時(shí)間：2018-10-22 09:35
【摘要】：內(nèi)皮祖細(xì)胞（endothelial progenitor cells, EPCs）分化在血管內(nèi)膜損傷修復(fù)和血管穩(wěn)態(tài)維持過(guò)程中均具有重要作用。血流產(chǎn)生的切應(yīng)力（shear stress）是調(diào)控EPC分化的關(guān)鍵因素之一，但其相關(guān)的力學(xué)生物學(xué)（mechanobiology）機(jī)制尚需進(jìn)一步闡明。 本文應(yīng)用平行平板流動(dòng)腔系統(tǒng)對(duì)人臍血來(lái)源的EPCs施加15dyn/cm2生理水平的層流切應(yīng)力，作用時(shí)間24h，首先證明了切應(yīng)力促進(jìn)EPCs向成熟的內(nèi)皮細(xì)胞（endothelial cells, ECs）分化，且抑制其向平滑肌細(xì)胞（smooth muscular cells, SMCs）分化。為了進(jìn)一步探討切應(yīng)力條件下EPC分化的相關(guān)力學(xué)生物學(xué)機(jī)制，我們開(kāi)展了以下2部分研究： 在第一部分，首先研究了SIRT1在切應(yīng)力調(diào)控EPC分化中的作用機(jī)制。結(jié)果顯示，切應(yīng)力明顯地誘導(dǎo)了EPCs的Akt磷酸化活性、SIRT1表達(dá)升高、組蛋白H3在賴氨酸9位點(diǎn)的乙酰化（ac-H3K9）降低，且三者依次在6、12和24h達(dá)到峰值。轉(zhuǎn)染SIRT1特異性siRNA下調(diào)了EPCs的EC標(biāo)志分子KDR、VE-cadherin、vWF和CD31的表達(dá)，促進(jìn)SMC標(biāo)志分子α-SMA和sm22α的表達(dá)，同時(shí)增強(qiáng)了組蛋白H3的乙�；�；而SIRT1激活劑白藜蘆醇（resveratrol）對(duì)EPC分化標(biāo)志表達(dá)和組蛋白H3乙�；饔玫慕Y(jié)果與之相反。用PI3K的抑制劑wortmannin孵育EPCs，抑制其EC標(biāo)志分子，卻促進(jìn)了其SMC標(biāo)志分子的表達(dá)，，并下調(diào)了SIRT1表達(dá)，增強(qiáng)了組蛋白H3的乙酰化。此外，體外基質(zhì)膠微管形成（tube formation）實(shí)驗(yàn)表明，SIRT1促進(jìn)EPCs微管狀結(jié)構(gòu)的形成與連接。上述結(jié)果提示，PI3K/Akt-SIRT1-ac-H3K9信號(hào)通路可能參與了切應(yīng)力誘導(dǎo)的EPCs向ECs的分化。 在第二部分，我們探討了microRNA-34a（miR-34a）在切應(yīng)力調(diào)控EPC分化中的可能分子機(jī)制。結(jié)果顯示，切應(yīng)力時(shí)間依賴地誘導(dǎo)EPCs的miR-34a表達(dá)，并在12h達(dá)到峰值；應(yīng)用3種靶基因預(yù)測(cè)數(shù)據(jù)庫(kù)預(yù)測(cè)提示，F(xiàn)OXJ2可能作為miR-34a的潛在靶基因參與細(xì)胞功能調(diào)控。雙熒光報(bào)告基因?qū)嶒?yàn)證明，F(xiàn)OXJ2是miR-34a的直接靶基因。然后，分別應(yīng)用miR-34a mimics和inhibitor刺激EPCs，進(jìn)一步驗(yàn)證了miR-34a對(duì)FOXJ2的負(fù)向調(diào)節(jié)；并且切應(yīng)力對(duì)FOXJ2的表達(dá)具有抑制作用。miR-34a正向調(diào)控EPCs的EC標(biāo)志分子KDR、VE-cadherin、vWF和CD31的表達(dá)而負(fù)向調(diào)控SMC標(biāo)志分子α-SMA、sm22α、calponin和smmhc的表達(dá)。之后，進(jìn)一步過(guò)表達(dá)或干擾FOXJ2，結(jié)果顯示，F(xiàn)OXJ2對(duì)EPC分化的作用與miR-34a的作用相反。此外，干擾FOXJ2促進(jìn)了EPCs在體外基質(zhì)膠的微管形成。上述結(jié)果提示，miR-34a可能通過(guò)負(fù)向調(diào)控其靶基因FOXJ2，參與了切應(yīng)力誘導(dǎo)的EPCs向ECs分化。 綜上所述，生理水平的層流切應(yīng)力促進(jìn)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號(hào)通路。這些研究結(jié)果對(duì)闡明EPCs響應(yīng)力學(xué)刺激的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制有重要意義，也為血管損傷修復(fù)和缺血性疾病的治療提供了新的力學(xué)生物學(xué)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 偽 -SMA and sm22 偽, 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 偽 -SMA,sm22 偽, 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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.01
本文編號(hào)：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 偽 -SMA and sm22 偽, 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 偽 -SMA,sm22 偽, 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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.01
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