BHK_(21)细胞的中间纤维-lamina-核骨架体系

以系列选择性抽提技术与显示细胞骨架的整装电镜技术为基础,应用免疫胶体金标记与蛋白质成份的双向电泳分析技术,研究了BHK_(21)细胞的中间纤维-lamina与核骨架(核基质)结构体系及其主要的蛋白成份。BHK_(21)细胞的中间纤维-lamina与核骨架是在结构上相互联系,贯穿于核与质的网络体系。中间纤维单丝直径为10nm,能很好地被抗波形蛋白抗体-金颗粒所标记,生化分析同样说明BHK_(21)细胞中间纤维的主要成份是波形蛋白(vimentin),其分子量为55KD,等电点为5.6。中间纤维网在胞质内呈极性分布,与lamina密切联结。BHK_(21)细胞的lamina能被抗lamin A与C的单克隆抗体-金颗粒标记。双向电泳分析证明,lamina含有三种蛋白成份,即lamin A,B,C,其分子最分别为68KD,70KD与62KD,lamin A,C等电点均为6.9—7.2,而lamin B偏酸,其等电点为5.8。BHK_(21)细胞核骨架纤维网也可以被清晰的显示,其蛋白成份较为复杂,在双向电泳谱上经常出现多个清晰的斑点,很可能含有肌动蛋白(actin)。298KD核基质蛋白的单克隆抗体-金颗粒能准确的标记核骨架纤维。

THE INTERMEDIATE FILAMENT-LAMINA-NUCLEAR MATRIX SYSTEM OF BHK-21 CELLS

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO_2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughtout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diametre. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina.Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there arc lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly pre sented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.