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培养的皮质星形胶质细胞中间丝和微管的组织及溶解性特性

The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture.

作者信息

Goetschy J F, Ulrich G, Aunis D, Ciesielski-Treska J

出版信息

J Neurocytol. 1986 Jun;15(3):375-87. doi: 10.1007/BF01611439.

Abstract

The organization of intermediate filaments (IF) and microtubules (MT) and the solubility of intermediate filament proteins and tubulin in astrocytes which develop from cerebral hemispheres of neonatal rats in culture were examined using immunocytochemical and immunochemical approaches. Results of immunocytochemical studies demonstrated that in flat astrocytes which develop after 3 weeks of culturing in serum-supplemented medium, the IF containing vimentin and glial fibrillary acidic protein (GFAP) are concentrated around the nucleus and dispersed in an irregular fashion throughout the cytoplasm. Astrocytes which develop in serum-free hormonally-defined medium irrespective of whether they are bipolar, multipolar or flattened, have IF organized as a fibrous network of filaments distributed from the nuclear regions to the cell periphery. Under both culture conditions, vimentin and GFAP are resistant to extraction with low salt buffer containing nonionic detergent, indicating that the different cytoplasmic distribution of IF is unrelated to the solubility properties of vimentin and GFAP. Double immunolabelling experiments with polyclonal antibody to GFAP and monoclonal antibody to each alpha-tubulin or beta-tubulin reveal an extensive codistribution and parallel organization of IF and MT in all morphological types of astrocytes studied. Stabilization of MT with taxol, or depolymerization of MT with colchicine, cause dramatic changes in the distribution of IF and inhibit the extension of astrocyte processes in response to dibutyryl cyclic AMP (dBcAMP). In early stages of treatment with dBcAMP, renewal of culture medium without dBcAMP produces a rapid and permanent retraction of astrocyte processes, whereas in later stages the processes only retract partially and are then restored and maintained for several days in the absence of dBcAMP. The retraction of processes is accompanied by changes of immunocytochemical staining of IF with antibody to GFAP, which appears more intense and diffuse. However, electrophoretic and immunoblot analyses of detergent-extracted proteins from parallel cultures demonstrate that neither the amount nor the solubility of GFAP and vimentin are changed. Detergent extraction in MT stabilizing conditions shows that a substantial proportion of tubulin in astrocytes cultured in serum-containing and serum-free media is assembled into MT, most of which depolymerize on treatment with low temperature and Ca2+. Following long exposure to dBcAMP the proportion of cold/Ca2+-stable MT increases. The results suggest that the IF of astrocytes in culture are dependent on MT with respect to their cytoplasmic distribution.(ABSTRACT TRUNCATED AT 400 WORDS)

摘要

运用免疫细胞化学和免疫化学方法,研究了新生大鼠大脑半球培养的星形胶质细胞中中间丝(IF)和微管(MT)的组织情况,以及中间丝蛋白和微管蛋白的溶解性。免疫细胞化学研究结果表明,在补充血清的培养基中培养3周后形成的扁平星形胶质细胞中,含有波形蛋白和胶质纤维酸性蛋白(GFAP)的中间丝集中在细胞核周围,并以不规则方式分散于整个细胞质中。在无血清激素限定培养基中形成的星形胶质细胞,无论其是双极、多极还是扁平状,其中间丝都组织成从核区到细胞周边分布的纤维状网络。在这两种培养条件下,波形蛋白和GFAP都能抵抗含非离子去污剂的低盐缓冲液的提取,这表明中间丝不同的细胞质分布与波形蛋白和GFAP的溶解性无关。用抗GFAP多克隆抗体和抗各α-微管蛋白或β-微管蛋白单克隆抗体进行的双重免疫标记实验显示,在所有研究的星形胶质细胞形态类型中,中间丝和微管广泛共分布且平行排列。用紫杉醇稳定微管,或用秋水仙碱使微管解聚,会导致中间丝分布发生显著变化,并抑制星形胶质细胞对二丁酰环磷腺苷(dBcAMP)作出反应时突起的延伸。在用dBcAMP处理的早期阶段,更换不含dBcAMP的培养基会使星形胶质细胞突起迅速且永久回缩,而在后期阶段,突起仅部分回缩,然后在无dBcAMP的情况下恢复并维持数天。突起的回缩伴随着用抗GFAP抗体对中间丝进行免疫细胞化学染色的变化,染色显得更强烈且弥散。然而,对平行培养物中去污剂提取蛋白的电泳和免疫印迹分析表明,GFAP和波形蛋白的量及溶解性均未改变。在微管稳定条件下进行去污剂提取显示,在含血清和无血清培养基中培养的星形胶质细胞中,相当一部分微管蛋白组装成微管,其中大部分在低温和Ca2+处理时解聚。长时间暴露于dBcAMP后,冷/Ca2+稳定微管的比例增加。结果表明,培养的星形胶质细胞的中间丝在细胞质分布方面依赖于微管。(摘要截选至400字)

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