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Investigation of the effects of a panel of IGFBP-5 mutants on apoptosis in a 3D mammary co-culture system

研究一组 IGFBP-5 突变体对 3D 乳腺共培养系统中细胞凋亡的影响

基本信息

批准号：
BB/F00205X/1
负责人：
David James Flint
金额：
$ 36.75万
依托单位：
University of Strathclyde
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FF00205X%2F1
关键词：
Investigation effects panel IGFBP mutants

项目摘要

The mammary gland develops as a rudimentary ductal structure in the mammary fat pad, during pre- and post-pubertal priods. During pregnancy it undergoes massive development resulting in differentiated epithelial cells which ultimately form the secretory alveolar structures that make milk during lactation. At the end of lactation the vast majority of the epithelial cells die by a process of programmed cell death known as apoptosis. The development and survival of the mammary gland depends upon a variety of hormones and growth factors, but it has been shown that the absence of insulin-like growth factor-I (IGF-I) leads to a dramatic impairment of mammary development. IGF-I is now known to be an important survival factor for many different cell types in the body. Surprisingly, the process of cell death at the end of lactation is not accompanied by a decrease in the concentration of IGF-I in the blood. Instead, we have demontrated that the epithelial cells produce a suicide protein, IGF-binding protein-5 (IGFBP-5) which binds to and inhibits the actions of IGF-I. The situation is actually more complex in that we, and others, have shown that IGFBP-5 can act independently of IGF-I, but the manner in which it does so is not yet understood. For example, IGFBP-5 can, independently of IGF-I , activate proteases which are involved in degrading the extracellular environment (a crucial part of the re-modeling of the mammary gland which occurs at the end of lactation). We have already succesfully generated mutated forms of IGFBP-5 in which the IGF-dependent and /independent effects have been separated. These molecules have been studied in preliminary fashion and shown to exhibit novel properties and a principal objective of the current proposal is to use these mutated forms of IGFBP-5 to provide a clearer insight into the mechanisms of action of IGFBP-5 in inducing mammary apoptosis and tissue remodelling. One of the strengths of this proposal lies in the use of a complex 3D model which more closely resembles the in vivo situation than do typical 2D cultures of cells. Our approach involves co-culture of mammary epithelial cells with fat cells (adipocytes) in a collagen/laminin-based extracellular environment. We believe this co-culture to be crucial as, firstly, the mammary epithelium develops as 3D ducts and alveolar structures which are polarised and have an internal cavity (processes which are not achieved with most cell lines). Secondly, the mammary epithelium interacts extensively with the mammary adipocytes, which secrete factors that influence and 'instruct' epithelial cell morphogenesis and differentiation. Our assesment of these co-cultures, and the effects of IGF-I and IGFBP-5 mutants therein, will involve state-of-the-art technologies including confocal microscopy, adenoviral infection with dominant-negative molecules and mutated proteins, and 96-well rtPCR screening approaches. We will also use more classical approaches of immnohistochemistry and western-immunoblotting techniques to examine intra-cellular signalling events. In addition, we will use rapid screening techniques, utilising established cell lines, in order to focus our studies of the more complex 3D cultures. Finally, we will take advantage of transgenic animals expressing the mutant IGFBP-5 molecules specifically in the mammary gland (provided by a separate project) to compare their phenotype with that induced by over-expression of non-mutated IGFBP-5 (this impairs mammary development in vivo). Thus these studies will identify, in vivo and in co-cultures, the relative importance of the IGF-dependent and IGF-independent effects of IGFBP-5 and explore whether this involves changes in cell surface proteins which influence cell survival and migration. We also aim to determine which intracellular signalling pathway(s) are activated by IGFBP-5.

在青春期前和青春期后，乳腺发育为乳腺脂肪垫的初级导管结构。在怀孕期间，它经历了大量的发育，导致分化的上皮细胞，最终形成分泌肺泡结构，在哺乳期产生乳汁。在哺乳结束时，绝大多数上皮细胞通过称为凋亡的程序性细胞死亡过程死亡。乳腺的发育和存活取决于多种激素和生长因子，但研究表明，缺乏胰岛素样生长因子- i （IGF-I）会导致乳腺发育的严重损害。现在已知igf - 1是体内许多不同细胞类型的重要存活因子。令人惊讶的是，哺乳结束时细胞死亡的过程并不伴随着血液中igf - 1浓度的降低。相反，我们已经证明上皮细胞产生一种自杀蛋白，igf结合蛋白-5 (IGFBP-5)，它与igf - 1结合并抑制其作用。情况实际上更复杂，因为我们和其他人已经表明，IGFBP-5可以独立于igf - 1起作用，但其作用方式尚不清楚。例如，IGFBP-5可以独立于igf - 1，激活参与降解细胞外环境的蛋白酶（在哺乳结束时发生的乳腺重塑的关键部分）。我们已经成功地生成了IGFBP-5的突变形式，其中igf依赖性和/非依赖性作用已经分离。这些分子已经进行了初步研究，并显示出新的特性，当前建议的主要目标是利用这些突变形式的IGFBP-5来更清楚地了解IGFBP-5在诱导乳腺细胞凋亡和组织重塑中的作用机制。这一建议的优势之一在于使用复杂的3D模型，它比典型的2D细胞培养更接近于体内情况。我们的方法包括在以胶原/层粘连蛋白为基础的细胞外环境中，将乳腺上皮细胞与脂肪细胞（脂肪细胞）共同培养。我们认为这种共培养是至关重要的，因为首先，乳腺上皮发育为三维导管和肺泡结构，这些结构极化并具有内腔（大多数细胞系无法实现的过程）。其次，乳腺上皮与乳腺脂肪细胞广泛相互作用，脂肪细胞分泌影响和“指导”上皮细胞形态发生和分化的因子。我们对这些共培养物的评估，以及其中的igf - 1和IGFBP-5突变体的影响，将涉及最先进的技术，包括共聚焦显微镜，显性阴性分子和突变蛋白的腺病毒感染，以及96孔rt - pcr筛选方法。我们还将使用更经典的免疫组织化学方法和western免疫印迹技术来检查细胞内信号事件。此外，我们将使用快速筛选技术，利用已建立的细胞系，以集中研究更复杂的3D培养。最后，我们将利用在乳腺中特异性表达突变IGFBP-5分子的转基因动物（由一个单独的项目提供）来比较它们的表型与过度表达非突变IGFBP-5诱导的表型（这会损害体内乳腺发育）。因此，这些研究将在体内和共培养中确定IGFBP-5的igf依赖性和igf非依赖性作用的相对重要性，并探讨这是否涉及影响细胞存活和迁移的细胞表面蛋白的变化。我们还旨在确定哪些细胞内信号通路被IGFBP-5激活。