P38alpha in Mouse Embryonic Stem Cells

描述(申请人提供)：p38丝裂原活化蛋白(MAP)激酶是广泛表达的蛋白激酶，调节生长和发育。P38α是哺乳动物细胞中表达最丰富的异构体。根据细胞类型和刺激的性质，p38α调节广泛的生理过程，如细胞增殖、存活和分化。它在胚胎发育中的关键作用在p38A基因敲除(P38A-/-)小鼠中得到了最好的证明，这些小鼠的胚胎表现出血管形成和血管结构缺陷，这表明P38Alpha在血管系统的发育中起着关键作用。P38α基因敲除是致命的，因此可以从动物模型中获得有限的信息。然而，从基因敲除的胚胎中分离的胚胎干细胞(ES细胞)(P38A-/-ES细胞)是有活力的，这可能是一个有价值的细胞系统来分析P38Alpha的特定功能。P38α在体细胞中已被广泛研究，但对其在ES细胞中的功能知之甚少。我们已经证明，P38A-/-ES细胞表现出与野生型ES细胞不同的几个特性，包括细胞黏附、形态和活性。本研究的目的是：1.进一步鉴定P38A和P38A-/-ES细胞系，探讨p38α缺失导致ES细胞特性改变的分子机制；2.研究p38α在ES细胞分化中的作用，特别是研究P38A-/-ES细胞向内皮细胞分化的能力；3.通过三维(3D)血管生成模型分析ES细胞分化为内皮细胞的功能。在这个模型中，内皮细胞将被培养在3D胶原基质中，在那里它们经历一系列的形态变化，形成管状结构，模仿体内血管形成的步骤。这一新的实验将被用作一种功能分析来检验一种假设，即来源于P38A-/-ES细胞的ECs可能保持一定的分化能力，但分化后的细胞可能具有组装成正常血管的功能受损。这项研究有望提供具有良好特性和基因定义的pSSalpha缺乏症ES细胞系，这将对深入研究pSSalpha在ES细胞分化和血管发育中的作用特别有用。从这项研究中获得的知识可能对于开发基于细胞的治疗方法来治疗与血管生成和心血管功能障碍相关的疾病有价值。

DESCRIPTION (provided by applicant): p38 mitogen activated protein (MAP) kinases are widely expressed protein kinases that regulate growth and development. p38alpha is the most abundant isoform expressed in mammalian cells. Depending on cell types and the nature of stimuli, p38alpha regulates a wide range of physiological processes, such as cell proliferation, survival, and differentiation. Its pivotal role in embryogenesis is best demonstrated in p38alpha knockout (p38a-/-) mice where embryos display defective vascularization and vessel structures, suggesting a critical role of p38alpha in the development of vascular system. p38alpha knockout is lethal, thus limited information can be obtained from animal models. However, the embryonic stem (ES) cells isolated from knockout embryos (p38a-/-ES cells) are viable, which can be a valuable cell system to analyze the specific functions of p38alpha. p38alpha has been intensively studied in somatic cells, but we know little of its functions in ES cells. We have shown that p38a-/-ES cells display several altered properties from wild type ES cells, including cell adhesion, morphology and viability. The goals of this proposal are: 1. to further characterize several lines of p38a and p38a-/-ES cells and to determine the molecular mechanisms underlying the altered properties of ES cells caused by p38alpha deletion, 2. to investigate the role of p38alpha in ES differentiation, specifically focusing on how the ability of ES cells to differentiate to endothelial cells (ECs) is affected in p38a-/-ES cell, and 3. to analyze ES cell-differentiated EC function by an 3-dimensional (3D) in vitro angiogenesis model. In this model, ECs will be cultured in a 3D collagen matrix, where they undergo a series of morphological changes to form tube-like structures, mimicking the steps of in vivo blood vessel formation. This novel assay will be used as a functional analysis to test hypothesis that ECs derived from p38a-/-ES cells may retain certain abilities to differentiate, but differentiated cells may have impaired functions to assemble into normal vessels. This study is expected to provide well characterized and genetically defined pSSalpha deficiency ES cell lines, which will be particularly useful for in-depth investigation of the roles of pSSalpha in ES cell differentiation and vascular development. The knowledge derived from this study could be valuable for the development of cell-based therapeutic approaches for diseases associated with angiogenesis and cardiovascular malfunction.