Dalby Matthew J, Yarwood Stephen J
Center for Cell Engineering, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Methods Mol Biol. 2007;370:121-34. doi: 10.1007/978-1-59745-353-0_10.
Focal adhesions and the cell cytoskeleton (intermediate filaments, microfilaments, microtubules) are involved in mechanotransduction-both direct (transduction of mechanical forces to the nucleus) and indirect (transduction of chemical signaling cascades to the nucleus). Thus, observation of changes in focal adhesion and cytoskeletal organization can be invaluable in research such as drug treatments and medical material testing in vitro. Here we describe how to stain human fibroblasts for vinculin (located to focal adhesions), actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments) and how to perform custom microarray experiments. Comparative analysis of the immunofluorescence and array data should allow the researcher to build up a global picture of changes to both direct and indirect mechanotransduction through the cytoskeleton from focal adhesions.
粘着斑和细胞骨架（中间丝、微丝、微管）参与机械转导——包括直接（将机械力转导至细胞核）和间接（将化学信号级联转导至细胞核）。因此，观察粘着斑和细胞骨架组织的变化在体外药物治疗和医用材料测试等研究中可能具有极高的价值。在此，我们描述如何对人成纤维细胞进行纽蛋白（定位于粘着斑）、肌动蛋白（微丝）、微管蛋白（微管）和波形蛋白（中间丝）的染色，以及如何进行定制微阵列实验。对免疫荧光和阵列数据的比较分析应能使研究人员全面了解通过细胞骨架从粘着斑进行的直接和间接机械转导的变化情况。
