切应力条件下核膜蛋白Nesprin2和LaminA在血管内皮细胞增殖和凋亡中的作用

发布时间：2018-04-21 13:41

  本文选题：切应力 + 血管重建　； 参考：《上海交通大学》2015年博士论文

【摘要】：血管重建(remodeling)是动脉粥样硬化和高血压等心血管疾病的发病基础,机械应力在其中起着重要的调控作用。血管内皮细胞(endothelial cells,ECs)和血管平滑肌细胞(vascular smooth muscle cells,VSMCs)是构成血管壁的主要细胞成分;ECs衬于血管壁内表面,直接受到血流经过所产生的流体切应力的作用;VSMCs与ECs在结构上相邻,在功能上相互影响。ECs受到流体切应力和相邻的VSMCs的共同作用,其稳态(homeostasis)在血管重建中有着重要意义。力学因素诱导血管重建的机制目前并未完全阐明,该机制的研究对于深入了解心血管疾病的发病基础及防治都具有重要意义。本实验室前期的血管组织差异蛋白质组学结果提示,核纤层蛋白Lamin A可能是力学敏感蛋白;因此,本文围绕核膜蛋白Nesprin2、SUN1和Lamin A展开了不同切应力条件下ECs增殖和凋亡变化的机制研究。本文应用平行平板流动腔系统,对单独培养的大鼠胸主动脉ECs施加15 dyn/cm2的正常切应力和5 dyn/cm2的低切应力,结果显示,低切应力明显抑制了核膜蛋白Nesprin2、SUN1和Lamin A的表达,并明显促进了ECs的增殖和凋亡;ECs单独培养、ECs与VSMCs接触联合培养以及ECs与VSMCs非接触联合培养3种不同的培养方式中,VSMCs对ECs核膜蛋白的表达并无显著影响。ECs分别转染Nesprin2、SUN1和Lamin A的特异性干扰片段后,ECs的核膜蛋白表达的减少均导致了ECs增殖和凋亡的异常增加。为了阐明核膜蛋白参与低切应力诱导ECs增殖和凋亡的机制,我们对Nesprin2和Lamin A蛋白进行了深入研究,分别构建了Nesprin2和Lamin A的过表达质粒,发现低切应力条件下ECs的Nesprin2和Lamin A的过表达能够逆转低切应力诱导的ECs增殖和凋亡。应用转录因子活性芯片技术,将ECs分别进行Nesprin2和Lamin A特异性干扰片段的转染和过表达质粒的转染并检测。芯片结果显示,Nesprin2表达水平的减少能够显著提高转录因子AP-2和TFIID的活性,而Lamin A表达水平的降低显著抑制了转录因子Stat-1、Stat-3、Stat-5和Stat-6的磷酸化。在静态干扰条件下和切应力加载条件下,再分别对芯片结果进行了验证。结果证实了上述转录因子在切应力调控Nesprin2和Lamin A变化中的作用。然后,应用Ingenuity Pathway Analysis(IPA)软件预测,得到了Nesprin2调控的转录因子AP-2的靶基因PLA2G16、PITX2、GEM、KISS1、KRT14和TFIID的靶基因FOS、LDLR和Gh,以及Lamin A调控的转录因子Stat-1、Stat-3、Stat-5和Stat-6的共同靶基因BCL2L1、CCND2、IRF1、IFNG、IL4和TNF。应用荧光定量PCR技术,我们找到了参与影响低切应力诱导ECs增殖和凋亡的靶基因,即AP-2的靶基因GEM、TFIID的靶基因FOS和LDLR以及Stat-1、Stat-3、Stat-5和Stat-6的共同靶基因BCL2L1、CCND2、IRF1、IFNG和IL4。综上所述,在低切应力条件下,ECs的核膜蛋白Nesprin2、SUN1和Lamin A的表达异常下降,其中外核膜蛋白Nesprin2表达下降后引起其下游转录因子AP-2和TFIID的表达上升,AP-2通过正调控其靶基因GEM,TFIID通过正调控其靶基因FOS并同时负调控靶基因LDLR,最终诱导ECs的增殖和凋亡。另一方面,低切应力抑制核纤层蛋白Lamin A表达,从而下调了Stat-1、Stat-3、Stat-5和Stat-6的磷酸化,Stat蛋白的磷酸化又通过正调控靶基因BCL2L1、IRF1、IFNG和IL4,并负调控CCND2,最终促进了ECs的增殖和凋亡。这些研究结果对阐明低切应力诱导ECs功能异常的力学生物学机制发挥了重要作用,并为动脉粥样硬化等心血管疾病的发病基础研究提供了新的力学生物学思路。
[Abstract]:Vascular reconstruction (remodeling) is the basis of cardiovascular diseases such as atherosclerosis and hypertension. Mechanical stress plays an important regulatory role. Vascular endothelial cells (endothelial cells, ECs) and vascular smooth muscle cells (vascular smooth muscle cells, VSMCs) are the main components of the vascular wall; ECs is lined with vascular wall. The inner surface is directly affected by the fluid shear stress produced by the flow of blood flow; VSMCs and ECs are adjacent to the structure, and the function of the interaction of.ECs to the interaction of the shear stress and the adjacent VSMCs, its steady-state (homeostasis) is of great significance in the reconstruction of blood vessels. The study of this mechanism is of great significance to the deep understanding of the basis and prevention of the pathogenesis of cardiovascular disease. The results of vascular tissue differential proteomics at the early stage of our laboratory suggest that the nuclear fibrinolytic protein Lamin A may be a mechanical sensitive protein; therefore, different shear stresses are expanded around the nuclear membrane protein Nesprin2, SUN1 and Lamin A. Study on the mechanism of ECs proliferation and apoptosis. In this paper, a parallel plate flow cavity system was used to exert 15 dyn/cm2 normal shear stress and 5 dyn/cm2 low shear stress on the isolated rat thoracic aorta, and the results showed that the low shear stress obviously inhibited the expression of the nuclear membrane protein Nesprin2, SUN1 and Lamin A, and obviously promoted ECs. Proliferation and apoptosis, ECs alone culture, ECs and VSMCs contact combined culture, and ECs and VSMCs non contact culture 3 different culture methods, VSMCs has no significant effect on the expression of ECs nuclear membrane protein. After.ECs transfected with Nesprin2, SUN1 and Lamin A, the decrease of the expression of nuclear membrane protein leads to the proliferation of.ECs. In order to elucidate the mechanism of the involvement of nuclear membrane protein in low shear stress induced ECs proliferation and apoptosis, we have conducted in-depth studies on Nesprin2 and Lamin A proteins and constructed Nesprin2 and Lamin A overexpressed plasmids respectively. It is found that the overexpression of ECs Nesprin2 and Lamin A can reverse low shear stress induced by low shear stress. ECs proliferation and apoptosis. Using transcription factor active chip technology, transfection of Nesprin2 and Lamin A specific interference fragments and transfection and detection of overexpressed plasmids respectively. The microchip results show that the decrease of Nesprin2 expression level can significantly increase the activity of AP-2 and TFIID of the transcription factor, and the expression level of Lamin A is decreased significantly. The phosphorylation of the transcription factors Stat-1, Stat-3, Stat-5 and Stat-6 was inhibited. The results of the chip were verified under static and shear stress loading conditions. The results confirmed the role of the above transcriptional factors in the change of Nesprin2 and Lamin A in the shear stress. Then, the Ingenuity Pathway Analysis (IPA) software was used. We found the target genes PLA2G16, PITX2, GEM, KISS1, KRT14 and TFIID, the target genes of the transcriptional factor AP-2, which are regulated by Nesprin2. The target genes that can induce the proliferation and apoptosis of ECs, namely, the target gene GEM of AP-2, the target genes of TFIID, FOS and LDLR, and the common target genes of Stat-1, Stat-3, Stat-5 and Stat-6, are abnormally decreased in the low shear stress conditions, including the outer nuclear membrane protein. When the expression of in2 decreased, the expression of the downstream transcription factor AP-2 and TFIID increased, and AP-2 regulated its target gene GEM, TFIID regulated its target gene FOS and negatively regulated the target gene LDLR, eventually inducing the proliferation and apoptosis of ECs. On the other hand, low shear stress inhibited the A expression of nuclear fibrinolytic Lamin, thus downregulating Stat-1. The phosphorylation of tat-5 and Stat-6, the phosphorylation of Stat protein, also regulates the target genes BCL2L1, IRF1, IFNG and IL4, and negatively regulates CCND2, which ultimately promotes the proliferation and apoptosis of ECs. These results play an important role in elucidating the mechanical biological mechanism of low shear stress induced ECs dysfunction, and for cardiovascular diseases such as atherosclerosis. The basic research of disease provides a new idea of mechanical biology.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R54

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