【摘要】 我们利用兔抗微管蛋白抗体和兔抗辣根过氧化物酶(HRP)抗血清制备的HRP—抗HRP(PAP)复合物,建立了微管的PAP免疫酶细胞化学方法。应用此法观察到人食管癌ECa 109、胃癌SGC 7901,乳腺癌MCF 7和成骨肉瘤OS 732细胞间期胞质微管减少或消失,只有大量弥散分布的微管蛋白棕色反应产物,在微管组织中心(MTOC)附近十分密集,而正常成纤维细胞和胎儿胃粘膜上皮细胞间期,都有发达的胞质微管结构(CMTC)。在分裂期,这些肿瘤细胞都显示纺锤体微管,与正常细胞比较无明显差异。本研究应用PAP方法进一步证明,以前用免疫荧光细胞化学方法观察到的人肿瘤细胞间期胞质微管缺陷的特征。除去低温(4℃)或秋水仙酰胺处理后,解聚的CMTC又可恢复,表明本方法与免疫荧光染色法,同样具有很高的特异性。本工作在细胞固定及免疫反应的某些步骤上有所改进。更多还原

【Abstract】 Peroxidase-Antiperoxidase (PAP) soluble complex was prepared and used in immunoperoxidase visualization of microtubules and microtubular proteins in human esophageal carcinoma ECa109, stomach cancer SGC7901, breast cancer MCF7 and osteosarcoma OS732 cell lines with human foreskin fibroblast cells and stomach epithelial cells as normal control. In comparison with the well-developed CMTC (Cytoplasmic Microtubule Complex) of normal cells, the cytoplasmic microtubules of tumor cells largely decreased in number and length or totally disappeared and replaced by brown precipitates of free non-microtubular tubulin components in the cytoplasm and more concentrated at the centrosomal region. The mitotic spindles of tumor cells were similar to those of normal cells. These results of PAP immunocytochemical method evidently confirmed our preliminary immunofluorescent observations on tumor cells which were considered to be microtubule-deficient during interphase. The CMTC pattern of stomach epithelial cells shown in this report be useful as a normal counterpart of tumor cells with epithelial origin is suggested. The cytoplasmic microtubules stained by PAP method were as sensitive to antimicrotubule factors such as low temperature or colcemid as were those stained by immunofluorescent method. Cytoplasmic microtubules underwent reversible depolymerization after low temperature or colcemid treatment and recovered again when released from the treatment. This is also a strong evidence for the efficiency and specificity of PAP method for microtubutes in our report Some modifications to De Mey’s PAP-microtubule method in our work were described, using chloroform: methanol (2:1) as fixatives, using 0.3％ Triton X100/PBS as anti-serum diluting solvent; using 1％ Triton X-100/PBS in washing steps, all were proved to be advantageous not only in preserving cell shape and microtubule structures but also in simplifying the staining procedures and saving time with equally good staining results to those in other reports.更多还原