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目的 探讨影响Ⅰ型IL 1受体 (IL 1RⅠ )转位至焦点附着斑的相关因素。方法 将含有绿色荧光蛋白 (GFP)基因的重组型和突变型 (C末端突变 )IL 1RⅠ质粒转染进入成纤维细胞 ,细胞生长于有或无纤维粘连蛋白的培养皿上 ,用激光共聚焦显微镜直接观察所表达的融合蛋白在细胞内的定位 ,IL 1β刺激后发生转位 ,并观察经免疫细胞化学染色后 ,细胞骨架蛋白Vinculin的位置。结果 转染了重组型IL 1R1质粒的细胞生长在纤维粘连蛋白上 ,表达的受体融合蛋白与Vinculin位于细胞膜的焦点附着斑 ,用IL 1β刺激可致受体转位至细胞内。转染突变型质粒的细胞 ,表达的融合蛋白位于胞质的核周区域 ,IL 1β刺激后不产生转位。 结论 Ⅰ型IL 1受体转位至焦点附着斑需要Vinculin等细胞外基质的参与并与受体C 末端保守结构域有关
Objective To investigate the factors that affect the translocation of type 1 IL 1 receptor (IL 1 RI) to focal attachment spots. Methods Recombinant and mutant (C-terminal mutant) IL1RⅠ plasmids containing green fluorescent protein (GFP) gene were transfected into fibroblasts, and the cells were cultured on Petri dishes with or without fibronectin. Directly observe the localization of the expressed fusion protein in cells, translocation after IL 1β stimulation, and observe the location of the cytoskeletal protein Vinculin after immunocytochemical staining. Results The cells transfected with the recombinant IL-1R1 plasmid grew on fibronectin. The expressed fusion protein fused with Vinculin at the focal spot of the cell membrane and stimulated by IL 1β to translocate the receptor into the cell. Transfected with mutant plasmid cells, the expression of the fusion protein located in the perinuclear region of the cytoplasm, IL 1β stimulation does not produce translocation. Conclusion The translocation of type I IL-1 receptors to focal attachment sites requires the participation of extracellular matrix such as Vinculin and is related to the C-terminal conserved domain of the receptor