Adipocyte dedifferentiation in the ceiling culture system: regulators of cell matrix remodelling

这项NSERC发现计划最初是在2011年资助的。2015年提交的延长提案获得了一年的资金。本申请提供了一个经修订的项目，其中考虑到了审查员提出的建设性意见。 我们的总体目标是利用天花板培养方法进一步表征成熟脂肪细胞向成纤维细胞样细胞的去分化过程，重点是这一现象的原因。拟议的实验是对赠款第一期所取得结果的直接和合乎逻辑的扩展。具体地说，下面介绍的项目遵循了我们开发的六孔平板、非低氧天花板培养方法和时间推移显微镜，以及我们对基质重塑因子(如转化生长因子(TGF)、成纤维细胞激活蛋白(FAP)和二肽基肽酶IV(DPP4)在脂肪细胞去分化过程中的潜在作用的发现。我们提出了四个具体目标，每个目标代表一个特定的项目。 具体目标#1：解决关于污染间充质细胞的问题的不确定性，该问题可能存在于天花板培养中经历去分化的分离的成熟脂肪细胞。我们将从一种在成熟脂肪细胞中特异表达GFP的小鼠品系中分离出成熟脂肪细胞，并在体外监测去分化情况。我们还将测试细胞悬浮液洗涤、细胞悬液离心法和细胞增殖抑制剂的影响。 特定目的#2：研究转化生长因子-α在脂肪细胞去分化过程中的作用。我们将从一种缺乏转化生长因子-1信号的小鼠品系中分离成熟脂肪细胞，并在体外监测细胞去分化。转化生长因子-1也将与药物抑制剂一起使用。 具体目的#3：研究FAP在脂肪细胞去分化过程中的作用。FAP的药理抑制剂以及从FAP缺陷小鼠分离的脂肪细胞将被用来建立和表征这种酶在天花板培养中去分化的重要性。 特定目的#4：研究DPP4在脂肪细胞去分化过程中的作用。DPP4的药理抑制剂以及从DPP4基因缺失的小鼠中分离的脂肪细胞将被用来建立和表征这种酶在天花板培养的脂肪细胞去分化中的重要性。 大多数致力于这一领域的研究小组主要关注脱分化过程中产生的细胞，而不是转化本身。我们认为，更好地了解这一过程的驱动因素或抑制因素，以及改进的培养技术，将极大地促进去分化脂肪细胞在组织工程领域的使用。所开发的技术将有助于HQP的训练，并为脂肪细胞生物学研究提供新的模型。

This NSERC Discovery program was initially funded in 2011. The renewal proposal submitted in 2015 was awarded one year of funding. The present application offers a revised project taking into account the constructive comments made by Reviewers. Our General Objective is to further characterize the dedifferentiation process of mature adipocytes toward fibroblast-like cells using the ceiling culture approach, with a focus on causal agents of this phenomenon. The experiments proposed represent a direct and logical extension of the results obtained in the first term of the grant. Specifically, the projects described below follow our development of a six-well plate, non-hypoxic ceiling culture approach and time-lapse microscopy, as well as our discoveries on the potential role of matrix-remodelling factors such as Transforming growth factor (TGF), Fibroblast-activation protein (FAP) and Dipeptidyl peptidase IV (DPP4) in the process of adipocyte dedifferentiation. We propose four Specific Aims, each representing a specific project. Specific Aim #1: To address uncertainty regarding the issue of contaminating mesenchymal cells that may be present with isolated mature adipocytes undergoing dedifferentiation in ceiling cultures. We will isolate mature adipocytes from a mouse strain overexpressing GFP specifically in mature adipocytes, and monitor dedifferentiation in vitro. We will also test the impact of cell suspension washing, cell suspension centrifugation and inhibitors of cell proliferation. Specific Aim #2: To characterize the role of TGF- in the process of adipocyte dedifferentiation. We will isolate mature adipocytes from a mouse strain deficient in TGF-1 signalling and monitor cell dedifferentiation in vitro. TGF-1 will also be used in conjunction with pharmacological inhibitors. Specific Aim #3: To characterize the role of FAP in the process of adipocyte dedifferentiation. Pharmacological inhibitors of FAP as well as adipocytes isolated from Fap-deficient mice will be used to establish and characterize the importance of this enzyme for dedifferentiation in ceiling cultures. Specific Aim #4: To characterize the role of DPP4 in the process of adipocyte dedifferentiation. Pharmacological inhibitors of DPP4 as well as adipocytes isolated from Dpp4-deificent mice will be used to establish and characterize the importance of this enzyme in adipocyte dedifferentiation in ceiling cultures. Most research groups working in this area focus mainly on the cells generated by the dedifferentiation process, not on the transformation itself. We propose that a better understanding of the drivers or inhibitors of this process, along with improved culture techniques, would greatly facilitate the use of dedifferentiated adipocytes in the tissue engineering realm. The techniques developed will contribute to HQP training and provide new models for studies on adipocyte biology.