Szabo-Pardi Thomas A, Agalave Nilesh M, Andrew Ashley T, Burton Michael D
School of Brain and Behavioral Science, Center for Advanced Pain Studies, University of Texas at Dallas.
School of Brain and Behavioral Science, Center for Advanced Pain Studies, University of Texas at Dallas;
J Vis Exp. 2019 Jul 11(149). doi: 10.3791/59647.
Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomato in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.
成纤维细胞是间充质细胞，激活后会改变其形态，最终影响它们所在组织的微环境。尽管传统成像技术有助于识别固定组织中的蛋白质相互作用和形态，但它们无法深入了解细胞结合和摄取蛋白质的速度有多快，以及一旦被激活，它们在体内的形态如何变化。在本研究中，我们提出两个主要问题：1）通过Toll样受体4（TLR4）和脂多糖（LPS）相互作用激活成纤维细胞的时间进程是怎样的？2）这些细胞一旦被激活会有怎样的行为？利用双光子成像技术，我们开发了一种新技术，用于评估LPS-FITC与其同源受体TLR4结合的能力，该受体在基因报告小鼠品系FSP1cre; tdTomato的外周成纤维细胞上表达。这种独特的方法使研究人员能够创建蛋白质与活细胞相互作用的深入、延时视频和/或图片，从而在理解蛋白质如何改变细胞行为方面有更高的精细度。
