CELL SORTER TO SUPPORT BASIC RESEARCH

这项建议的申请者在生理学、新陈代谢和细胞信号传递领域进行前沿的基础血脂研究。到目前为止，研究已经解决了脂肪酸、磷脂、机械压力和运动如何影响全身生理、营养新陈代谢和细胞信号的问题。这些基本问题已经通过整体培养、细胞培养和组织培养相结合的方法加以解决。人体内的绝大多数组织由多种类型的细胞组成。这使得很难确定给定组织床内的哪些特定细胞对干预有反应。研究基因和蛋白质变化的组织分析建立了所有分离的细胞都是相同的基本假设，然而，这是一个严重的过于简单化，也存在大量其他类型的细胞。例如，这些细胞可能包括间质和上皮细胞或浸润性免疫细胞。离心法和密度依赖法是可行的，但缺乏特异性和敏感性，因此需要大量的组织(即汇集多个动物的器官)才能获得相对不纯的制剂。同样，在细胞培养方法中，通常希望根据治疗后的表型特征对细胞进行分类，然后评估所需的实验终点。这些实验需求只能使用配备了细胞分选器的流式细胞仪来实现，而申请者并不容易获得这种基础设施。符合研究人员需求且价格具有竞争力的仪器是Bio-Rad S3e细胞分选仪。这是一种入门级的主力流式细胞仪，也具有分选能力。唯一的直接竞争对手是索尼SH800。然而，尽管在功能上与之相当，但它的价格并不具有竞争力。更高端的流式细胞仪的能力远远超出了这些研究人员的常规需求，而且成本也在100万美元以上。预计购买该仪器将加强对4个实验室的25名以上高素质人员的直接培训(每年)。此外，一旦圭尔夫大学人类健康和营养科学系设立，预计将有更多的教员及其受训人员寻求使用这一仪器。认识到这一文书的重要性，该部将提供财政支助，以满足预算需要。总体而言，分离纯化的细胞群体的能力将有助于阐明作用机制，并改善许多高素质人员的培训环境。

The applicants of this proposal conduct leading edge basic lipid research in fields of physiology, metabolism and cell signalling. Studies to date have addressed questions about how fatty acids, phospholipids, mechanical stress and exercise influence whole body physiology, nutrient metabolism and cell signalling. These fundamental questions have been addressed using a combination of whole body, cell and tissue culture approaches. The vast majority of tissues in the body are comprised of a wide range of cell types. This makes it difficult to determine which particular cells within a given tissue bed are responding to the intervention. Tissue analyses examining gene and protein changes make the basic assumption that all cells isolated are the same, however, this is a gross oversimplification and a multitude of other cell types are also present. For example, these may include stromal and epithelial cells or infiltrating immune cells. Centrifugation and density dependent methods are available but lack specificity and sensitivity, thus requiring large amounts of tissues (i.e. pooling organs from multiple animals) to obtain relatively impure preparations. Similarly, in cell culture approaches it is often desirable to sort cells based on phenotypical characteristics following treatment and then assess the desired experimental endpoints. These experimental needs can only be accomplished using a flow cytometer equipped with a cell sorter, infrastructure that is not readily available to the applicants. An instrument that meets the needs of the investigators and competitively priced is the Bio-Rad S3e cell sorter. This is an entry level workhorse flow cytometer which also has sorting capability. The only other direct competitor is the Sony SH800. However, while comparable in features, it is not competitively priced. Higher end flow cytometers have capabilities far exceeding the routine needs of these investigators and also costing upwards of 1 million. It is anticipated that acquiring this instrument will enhance the direct training of 25+ high quality personnel (per year) across 4 laboratories. Furthermore, it is anticipated that additional faculty and their trainees will seek the use of this instrument once established in the Department of Human Health and Nutritional Sciences at the University of Guelph. In recognition of the importance of this instrument, the Department will provide financial support to support budgetary needs. Overall, the ability to isolate purified populations of cells will enable the elucidation of mechanisms of action and enhance the training environment for many high quality personnel.