血流剪切力下人脐静脉内皮细胞生物学特性的研究

发布时间：2018-02-12 08:27

本文关键词： 内皮细胞回馈调节机械力（血流剪切力） microRNA PI3Kinase/MAP Kinase　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：心血管疾病发生发展的研究和microRNA的研究同属于21世纪生命科学的热点领域。要对心血管疾病，尤其是动脉粥状硬化的形成有全面和深入的认识，必须在整体、动态和网络的水平上对其进行研究。本实验以人脐静脉内皮细胞（HUVECs）为研究对象，应用体外血流剪切力刺激，对其信号通路、基因调控、生物学特性的改变，尤其是microRNA表达量的变化进行研究。对血流剪切力下HUVEC中的microRNA和信号通路之间回馈调节进行探索。采用灌注酶消化法分离所得的脐静脉内皮细胞不仅纯度高而且数量充足，细胞活力高、增殖能力强，通过鉴定符合血管内皮细胞的特性并且能够稳定的进行传代培养以及冻存复苏，所以能够很好的满足接下来的实验需要，对进一步研究血管内皮细胞的生物学特性有重要意义。应用体外血流动力学系统研究在剪切力下HUVECs的生物学特性的改变，首次发现了miR-19a和miR-23b/27b分别受PI3K和MAPK的调控，PI3K和MAPK可能分别为miR-19a和miR-23b/27b的上游调控因子。在用Antagomir-19a（AM19a）抑制miR-19a后，，PI3K通路被阻断；用AM23b、27b分别抑制miR-23b、27b后，MAPK通路中的三个成员ERK，P38，JNK的磷酸化全部下降，以上可以作为microRNA回馈调节信号通路的一个指示。而且过表达miR-19a可以逆转PI3K抑制剂造成的抑制效果；过表达miR-23b、27b对MAPK的活性有着类似的效果。因此，以上发现可证明PI3K和MAPK介导的剪切力调控的miR表达量转而又调控剪切力相关信号通路，在miR和信号通路之间存在着一个回馈调节的作用。综上所述，本研究以体外分离培养的HUVECs为研究对象明确了在血流剪切力下HUVECs的生物学特性变化；首次发现miR-19a与PI3K信号通路；miR-23b、27b与MAPK信号通路之间的回馈调节作用，为探索血流剪切力作用下HUVECs内信号通路和microRNA作用途径提供了理论基础，为更好地研究心血管病发生发展机理提供了充分的实验依据。
[Abstract]:In 21th century, the research on the occurrence and development of cardiovascular disease and the study of microRNA belong to the hot field of life science. In order to have a comprehensive and in-depth understanding of cardiovascular disease, especially the formation of atherosclerosis, it is necessary to have an overall understanding. In this study, human umbilical vein endothelial cells (HUVECs) were used to stimulate human umbilical vein endothelial cells (HUVECs) by external blood flow shear stress (ECF), and to change the signal pathway, gene regulation and biological characteristics of HUVECs. In particular, the change of microRNA expression was studied, and the feedback regulation between microRNA and signal pathway in HUVEC under shear stress was explored. The umbilical vein endothelial cells isolated by perfusion enzyme digestion not only have high purity and sufficient quantity, but also have high cell viability and strong proliferative ability. Through the identification of the characteristics of vascular endothelial cells and stable passage culture and cryopreservation and resuscitation, it can meet the needs of the following experiments, which is of great significance to further study the biological characteristics of vascular endothelial cells. The effects of shear stress on the biological characteristics of HUVECs were studied by using the extracorporeal hemodynamic system. It was found for the first time that miR-19a and miR-23b/27b were regulated by PI3K and MAPK respectively. PI3K and MAPK may be upstream regulatory factors of miR-19a and miR-23b/27b, respectively. The PI3K pathway was blocked after miR-19a was inhibited by Antagomir-19aA1a, and the phosphorylation of P38 JNK of three members of ERK P38JNK was inhibited by AM23bm27b, respectively. These can be used as an indication of the signal pathway of microRNA feedback regulation. Furthermore, overexpression of miR-19a can reverse the inhibitory effect of PI3K inhibitor, and overexpression of miR-23bf27b has a similar effect on the activity of MAPK. These findings suggest that the shear-stress regulated miR expression mediated by PI3K and MAPK in turn regulates the shear-stress-related signaling pathway, and there is a feedback regulation between miR and the signaling pathway. In conclusion, the biological characteristics of HUVECs under shear stress were determined by using isolated and cultured HUVECs in vitro, and the feedback regulation between miR-19a and PI3K signaling pathway miR-23bf27b and MAPK signaling pathway was first found. It provides a theoretical basis for exploring the signal pathway and microRNA action pathway in HUVECs under the action of blood flow shear stress, and provides a sufficient experimental basis for better studying the mechanism of occurrence and development of cardiovascular disease.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.01

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