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目的 :为进一步了解IL - 1受体的结构与IL - 1受体在细胞内表达后的转运的关系 ,以及该结构改变对IL - 1受体功能的影响。方法 :将重组型、突变型和仅含细胞外段的I型IL - 1受体 (IL - 1RI)的质粒构建入含有绿色荧光蛋白的表达载体中 ,用磷酸钙沉淀法转染入成纤维细胞中 ,通过激光扫描共聚焦显微镜 ,观察其表达的融合蛋白在单个活细胞中的定位 ,以及与IL - 1结合后的运动变化。结果 :重组型IL - 1RI的融合蛋白表达后主要位于细胞膜和细胞伸出的突起与细胞基质相接触的焦点附着斑处 ,加入IL - 1后 ,逐渐从细胞膜上移至细胞质内 ,最后聚集在细胞核上。突变型IL - 1RI和细胞外段IL - 1RI的融合蛋白表达后散布于细胞质内 ,对IL - 1的剌激不产生反应。结论 :纤维粘连蛋白可增加IL - 1RI的表达转染率。IL - 1RI表达后的转运过程与IL - 1RI细胞内段结构域的完整和精确有关 ,尤其需要C -末端第 5 14位色氨酸的参与。
OBJECTIVE: To further understand the relationship between the structure of IL - 1 receptor and the translocation of IL - 1 receptor after intracellular expression and the effect of the structural alteration on IL - 1 receptor function. Methods: Plasmids of recombinant, mutant and IL - 1 receptor (IL - 1RI) containing extracellular domain were constructed into expression vector containing green fluorescent protein and transfected into fibroblasts by calcium phosphate precipitation Cells were observed by laser scanning confocal microscopy, the localization of the expressed fusion protein in a single living cell, and the change of motility after IL - 1 binding. Results: The expression of recombinant IL - 1RI fusion protein was mainly located in the focal attachment spots where the cell protuberance and cell protuberance contacted with the cell matrix. After the addition of IL - 1, the fusion protein gradually migrated from the cell membrane to the cytoplasm and eventually accumulated in On the nucleus. The fusion protein of mutant IL - 1RI and extracellular domain IL - 1RI was expressed in the cytoplasm and did not respond to the stimulation of IL - 1. Conclusion: Fibronectin can increase the transfection efficiency of IL - 1RI. The translocation of IL - 1RI expression is related to the integrity and precision of the IL - 1RI intracellular domain, especially the involvement of tryptophan at the C - terminus.