Ezzell R M, Toner M, Hendricks K, Dunn J C, Tompkins R G, Yarmush M L
Surgical Services, Massachusetts General Hospital, Boston 02114.
Exp Cell Res. 1993 Oct;208(2):442-52. doi: 10.1006/excr.1993.1266.
The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.
细胞骨架对于维持多种细胞类型（包括肝细胞）的细胞形态和分化功能很重要。在本研究中，将夹在两层胶原凝胶之间的成年大鼠肝细胞与培养在单层胶原凝胶上的细胞进行比较，以观察细胞骨架蛋白肌动蛋白和微管蛋白在组织和表达上的差异。培养在两层水合大鼠尾腱胶原（夹心凝胶）之间的肝细胞在形态上与完整肝脏中的细胞相似，持续数周。这些肝细胞中的肌动蛋白丝（F-肌动蛋白）集中在细胞-细胞接触区域的质膜下方。相比之下，培养在单层胶原凝胶上的肝细胞扁平且具有运动性，并有含F-肌动蛋白的应力纤维。这伴随着肌动蛋白mRNA增加了几倍。微管在夹心凝胶中培养的肝细胞中形成交织网络，但在单层凝胶培养物中它们形成延伸至细胞周边的长平行阵列。在单层凝胶上培养的肝细胞中微管蛋白mRNA含量高出几倍。纤连蛋白和层粘连蛋白染色在单层凝胶培养物中更强，并且这些蛋白质集中在从细胞周边辐射出的原纤维中。在单层凝胶上培养的肝细胞上覆盖第二层胶原凝胶（双层凝胶挽救）可逆转细胞铺展并减少应力纤维。双层凝胶挽救还导致肌动蛋白和微管蛋白mRNA水平降低至夹心凝胶培养物和新鲜分离的肝细胞中的水平。这些结果表明，细胞外基质的构型对培养的大鼠肝细胞中的细胞骨架蛋白具有动态影响。
