RACK1调控的蛋白激酶家族在切应力诱导血管细胞增殖中的作用

发布时间：2018-03-11 05:04

  本文选题：低切应力　切入点：RACK1　出处：《上海交通大学》2014年硕士论文　论文类型：学位论文

【摘要】：心血管疾病，如动脉粥样硬化等给人类的生命健康带来了重大的危害。各种病理因素导致的血管重建(Vascular remodeling)是这类疾病共有的发病基础和基本的病理过程。 在体条件下的心血管系统，处于复杂的力学环境中，其中血流动力学是影响血管重建的重要因素。研究表明，，在血管分叉起始处和弯曲处容易形成动脉粥样硬化，在这些区域主要存在的是低切应力（lowshear stress, LowSS），揭示LowSS在血管重建的发生和发展中有着重要的作用，然而其中的力学生物学机制还没有被完全揭示。本实验室前期发现，在体外应力培养的血管组织中，LowSS条件下，活化激酶C受体1（receptor for activated C kinase1, RACK1）的表达明显升高，RACK1做为重要的蛋白激酶（protein kinase, PK）活性调控因子，其在切应力调控血管重建中的作用机制仍不明了。 基于此，本文对RACK1在血管细胞中的表达进行了验证，并探讨其在切应力调控血管内皮细胞（endothelial cells, ECs）和血管平滑肌细胞（vascular smooth muscle cells, VSMCs）增殖中的作用。本文运用联合培养模型，培养大鼠胸主动脉ECs和VSMCs，应用平行平板流动腔系统，分别施加15dyn/cm2的正常切应力（normal shear stress,NSS）和5dyn/cm2的LowSS，加载时间为12h；然后用ELISA方法检测细胞的增殖，应用Western blotting技术检测RACK1表达及PKCα/βII、PKD、Akt磷酸化的变化。之后，在静态条件下，运用RNA干扰方法，抑制ECs和VSMCs的RACK1表达，用BrdU ELISA方法检测细胞的增殖，采用Western blotting技术检测RACK1表达及PKCα/βII、PKD916、PKD744和Akt磷酸化的变化。 结果显示：①与NSS相比，LowSS上调了ECs与VSMCs的增殖水平。②与NSS相比，在LowSS条件下，ECs与VSMCs中RACK1的表达水平，Akt、PKCα/βII的磷酸化显著上调，同时LowSS显著上调了PKD916位点丝氨酸残基磷酸化，但对744位点丝氨酸残基磷酸化无明显作用。③静态条件下，抑制ECs和VSMCs的RACK1表达引起细胞增殖下调。④静态条件下，抑制ECs和VSMCs的RACK1表达，使PKC α/βII、PKD916、Akt的磷酸化水平下调。 结果表明，LowSS可诱导ECs和VSMCs的RACK1表达，从而活化PKCα/βII、PKD916、 Akt等多种细胞内信号分子，在血管重建时期促进细胞的增殖。
[Abstract]:Cardiovascular diseases, such as atherosclerosis, have brought great harm to human life and health. Vascular remodeling caused by various pathological factors is the common pathogenesis and basic pathological process of these diseases. In vivo, the cardiovascular system is in a complex mechanical environment, in which hemodynamics is an important factor affecting vascular remodeling. In these regions, low shear stress lowshear (LowSS) reveals that LowSS plays an important role in the occurrence and development of vascular remodeling, but the mechanism of mechanics and biology has not been fully revealed. Under the condition of LowSS, the expression of activated kinase C receptor for activated C kinase 1 (rack1) was significantly increased as an important regulatory factor of protein kinase protein kinase (PKK) activity. The mechanism of its role in the regulation of vascular remodeling by shear stress is still unclear. Based on this, the expression of RACK1 in vascular cells was verified, and the role of RACK1 in regulating the proliferation of vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMC) in vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMC) was investigated. ECs and VSMC were cultured in rat thoracic aorta. The normal shear stress shear (NSSs) of 15 dyn / cm 2 and low SS of 5 dyn / cm 2 were applied in parallel plate flow chamber system. The loading time was 12 h. Then the proliferation of cells was detected by ELISA method. Western blotting technique was used to detect the expression of RACK1 and the phosphorylation of PKC 伪 / 尾 PKC 伪 / 尾 PKDX. Then, under static condition, the RACK1 expression of ECs and VSMCs was inhibited by RNA interference method, and the proliferation of ECs and VSMCs was detected by BrdU ELISA method. The expression of RACK1 and the phosphorylation of PKC 伪 / 尾 -PKD916PKD744 and Akt were detected by Western blotting technique. The results showed that low SS upregulated the proliferation level of ECs and VSMCs. 2 compared with NSS, the expression level of RACK1 in LowSS and VSMCs was significantly up-regulated, and LowSS significantly up-regulated the phosphorylation of serine residues at PKD916 site. But there was no obvious effect on 744 site serine residue phosphorylation. Under static condition, inhibition of RACK1 expression of ECs and VSMCs resulted in down-regulation of cell proliferation under static condition, inhibition of RACK1 expression of ECs and VSMCs, and down-regulation of phosphorylation level of PKC 伪 / 尾 II-PKD916 / Akt. The results showed that low SS could induce the expression of RACK1 in ECs and VSMCs, thus activating many signal molecules such as PKC 伪 / 尾 II-PKD916, Akt and so on, which could promote the proliferation of cells during vascular reconstruction.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.01

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