Sawa H, Takeshita I, Fukui M
Department of Neuropathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Anticancer Res. 1989 Nov-Dec;9(6):1673-9.
We have previously described how mouse monoclonal antibody Mab S-11E10 specifically stained ruffling membranes and filopodia of cultured human glioma cells. We found that this antibody stains ruffling membranes, not only of human astrocytes and glioma cells, but also of rat astrocytes and rat C6 glioma cells. In addition, we newly prepared rabbit polyclonal antibody for the antigens, which were eluted by Mab S-11E10 affigel 10 (Bio Rad) affinity column chromatography from cultured rat C6 glioma cells. The polyclonal antibody detects 65KDa, 57KDa, 46KDa, 43KDa bands in Western blotting analysis and specifically stained ruffling membranes of human glioma cells on cell spreading. Using the polyclonal antibody, we studied the change of the distribution of the antigens during cell spreading and in fully spread cells. At the beginning of cell spreading, ruffling membranes were specifically stained, but 72 hours after plating the immunofluorescence was localized in membranes of supranuclear regions and small protuberances. Furthermore, we semi-quantitatively measured the contents of the antigens detected by the polyclonal antibody, using dot blotting immunoassay. The contents of the antigens rapidly increased at the beginning of cell spreading and thereafter gradually reduced. The change of the contents seemed to be time--and cell density--dependent and to relate to the extent of cell spreading. These results suggest that the antigens may play an important role in cell spreading of glial cells.
我们之前曾描述过小鼠单克隆抗体Mab S-11E10如何特异性地染色培养的人胶质瘤细胞的皱襞膜和丝状伪足。我们发现这种抗体不仅能染色人星形胶质细胞和胶质瘤细胞的皱襞膜，还能染色大鼠星形胶质细胞和大鼠C6胶质瘤细胞的皱襞膜。此外，我们新制备了针对这些抗原的兔多克隆抗体，这些抗原是通过Mab S-11E10琼脂糖凝胶10（伯乐公司）亲和柱层析从培养的大鼠C6胶质瘤细胞中洗脱得到的。该多克隆抗体在蛋白质免疫印迹分析中能检测到65KDa、57KDa、46KDa、43KDa的条带，并且在细胞铺展时能特异性地染色人胶质瘤细胞的皱襞膜。利用该多克隆抗体，我们研究了细胞铺展过程中和完全铺展细胞中抗原分布的变化。在细胞铺展开始时，皱襞膜被特异性染色，但接种后72小时，免疫荧光定位于核上区域的膜和小突起上。此外，我们使用斑点印迹免疫测定法半定量地测量了多克隆抗体检测到的抗原含量。抗原含量在细胞铺展开始时迅速增加，此后逐渐降低。这种含量的变化似乎与时间和细胞密度有关，并且与细胞铺展的程度有关。这些结果表明这些抗原可能在神经胶质细胞的细胞铺展中起重要作用。
