Tracy R P, Wold L E, Currie R M, Young D S
Clin Chem. 1982 Apr;28(4 Pt 2):915-9.
A two-dimensional gel "map" of the proteins in normal cells and transformed cells may well help establish the presence of putative tumor-specific protein antigens, which can be isolated and used as antigens. We so examined normal colon mucosa and colon adenocarcinoma, using a sensitive silver stain. Samples were promptly prepared after excision by rapidly trimming the tissue to a small, grossly homogeneous piece, which was frozen on solid CO2. Frozen histological sections and permanent mounts were also prepared for several areas, to document the cellular constitution and to grade the carcinoma. Sample preparation for electrophoresis is described. The DNA was pelleted by centrifugation (108 000 x g x 1.5 h). Little or no pellet was observed, indicating complete solubilization. Many of the tumors contained proteases, liberated during sample preparation, so protease inhibitors were included. A highly reproducible pattern was observed for normal mucosa. Comparison of the gels from paired samples with histological data allows many of the spots to be tentatively assigned to the different cell types present in the mucosa (epithelium, connective tissue, or muscularis mucosa) and, in most tumors analyzed, detection of a series of spots that are not detectable in the pattern for normal tissue. These apparently tumor-associated proteins may be of epithelial cell or stromal origin.
正常细胞和转化细胞中蛋白质的二维凝胶“图谱”很可能有助于确定假定的肿瘤特异性蛋白质抗原的存在，这些抗原可以被分离出来并用作抗原。我们使用灵敏的银染法对正常结肠黏膜和结肠腺癌进行了这样的检查。切除组织后，立即将其快速修剪成小块大体均匀的组织块，并在固态二氧化碳上冷冻，从而迅速制备样品。还对几个区域制备了冷冻组织切片和永久装片，以记录细胞构成并对癌进行分级。文中描述了用于电泳的样品制备方法。通过离心（108000×g×1.5小时）使DNA沉淀。几乎未观察到沉淀，表明已完全溶解。许多肿瘤在样品制备过程中释放出蛋白酶，因此加入了蛋白酶抑制剂。正常黏膜呈现出高度可重复的图谱。将配对样品的凝胶图谱与组织学数据进行比较，可以初步将许多斑点归属于黏膜中存在的不同细胞类型（上皮细胞、结缔组织或黏膜肌层），并且在大多数分析的肿瘤中，检测到一系列在正常组织图谱中无法检测到的斑点。这些明显与肿瘤相关的蛋白质可能来源于上皮细胞或基质。
