Kurokawa Y, Oguma K, Yokosawa N, Syuto B, Fukatsu R, Yamashita I
Department of Psychiatry and Neurology, School of Medicine, Hokkaido University, Sapporo, Japan.
J Gen Microbiol. 1987 Sep;133(9):2647-57. doi: 10.1099/00221287-133-9-2647.
Binding of purified Clostridium botulinum type A, C1 and E toxins to cultured cells was studied by an immunocytochemical method. Type A and C1 toxins bound strongly to neuron cultures prepared from brains of foetal mice, but binding of type E toxin was weak. None of the toxin types bound to the feeder layer, composed of non-neuronal cells. The heavy-chain component of the type C1 toxin bound to neurons, but the light chain component did not. Type C1 toxin also bound only to cell lines of neuronal origin. When type C1 toxin [final concentration 4 x 10(2) LD50 (10 ng) per well] was added to primary neuron cultures in 96-well plates, degeneration of neuronal processes and rounding of neuronal somas were observed, but type A and E toxins did not produce such changes. The binding and cytotoxic activities of type C1 toxin were blocked by heat treatment (80 degrees C for 30 min) or by preincubation of the toxin with polyclonal anti-C1 IgG and some of the monoclonal antibodies which neutralized the toxin activity in mice. In the neuronal processes treated with C1 toxin, many degenerated mitochondria, membranous dense bodies and vesicles were observed by electron microscopy; these ultrastructural changes were similar to those of Wallerian degeneration in vivo.
采用免疫细胞化学方法研究了纯化的A型、C1型和E型肉毒梭菌毒素与培养细胞的结合情况。A型和C1型毒素与从胎鼠脑制备的神经元培养物强烈结合，但E型毒素的结合较弱。没有一种毒素类型与由非神经元细胞组成的饲养层结合。C1型毒素的重链成分与神经元结合，但轻链成分不结合。C1型毒素也仅与神经元来源的细胞系结合。当将C1型毒素[每孔终浓度为4×10(2) LD50（10 ng）]添加到96孔板中的原代神经元培养物中时，观察到神经元突起的退化和神经元胞体的变圆，但A型和E型毒素未产生此类变化。C1型毒素的结合和细胞毒性活性可通过热处理（80℃ 30分钟）或通过将毒素与多克隆抗C1 IgG和一些在小鼠中中和毒素活性的单克隆抗体预孵育来阻断。在用C1型毒素处理的神经元突起中，通过电子显微镜观察到许多退化的线粒体、膜性致密体和囊泡；这些超微结构变化与体内华勒氏变性的变化相似。
