Involvement of Membrane Skeletal Proteins in the Stimulus-Response Coupling of the Neutrophil and Its Molecular Mechanism

1.包埋前免疫电子显微镜观察发现，正常人中性粒细胞胞膜及其周围100 nm范围内的大多数(&gt；75%)Fodrin不在细胞膜上，而是在细胞质内的丝状胞质内。这一结果不同于红细胞中含有speclrin和淋巴细胞中含有fodrin的结果，在这些结果中，大部分免疫金标记物都附着在细胞膜上。结果表明，中性粒细胞中的丝蛋白是细胞骨架蛋白，而不是外周膜Drotein。经趋化肽f-Met-Leu-Phe处理后，中性粒细胞形态发生分化，沿前尾轴方向出现明显的形态分化，其胞浆中的Fodrin主要集中在胞浆的后部；在室温条件下，胞浆中Fodrin呈极化分布的细胞比例在2min时达到70%以上，在10min时达到最大值，约为80%，之后逐渐下降。这种剧烈的重新分布发生得比覆盖淋巴细胞中的Fodrin的类似重新分布要快得多，后者在37ﾟC.3时通常需要10分钟以上。去除贴壁的中性粒细胞的上细胞膜，剩余的细胞的基底半部分用免疫金电镜法标记(去顶法)。未受刺激的细胞膜上免疫标记均匀，偶见直径5 nm的细丝被标记，经趋化肽刺激后，标记集中在细胞后半部，多束10 nm细丝平行排列。结果表明，胡芦素参与了中性粒细胞内细胞骨架成分的重新分布。在表皮和角膜上皮中也观察到类似的动态再分布。将去顶方法应用于嗜铬细胞，发现直径为2-3 nm的纤维被标记为Fodrin。

1. By preembedding immunoelectron microscopy, most(>75%)of fodrin in the normal human neutrophil was not seen on the cell membrane nor within 100 nm from it, but in the filamenlous cytoplasm further inside. This result was distinct from those with speclrin in the erythrocyte and fodrin in the lymphocyte, in which a majority of immunogold labeling was attached to the cell membrane. The result indicates that fodrin in the neutrophil is a cytoskeletal protein rather than a peripheral membrane Drotein.2. Neutrophils treated with a chemotactic peptide, f-Met-Leu-Phe, became morphologically differential"led along the front-tail axis, and fodrin became concentrated in the posterior region ; the percentage of cells showing the polarized distribution of fodrin was more than 70% at 2 min, reached the maximum of around 80% at 10 min at room temperature, and decreased thereafter. The drastic redistribution occurs much faster than the similar relocation of fodrin in capping lymphocytes, which generally takes more than 10 min at 37ﾟC.3. The superior cell membrane of adherent neutrophils was removed and the remaining basal half of the cells was labeled for immunogold electron microscopy(unroofing method). In unstimulated cells, immunolabeling was observed uniformly in the membrane and filaments of 5 nm in diameter were labeled occasionally ; after stimulation with the chemotactic peptide, labeling was concentrated in the posterior half of the cell where numerous bundles of 10 nm filaments run in parallel. The result indicates that fodrin mediates'the redistribution of cytoskeletal components in the neutrophil.4. Similar dynamic redistribution was observed in the epidermis and the corneal epithelium. The unroofing method was soplied to the chromaffin cell and filaments of 2-3 nm in diameter were found labeled for fodrin.