Vegunta R K, Cooney D R, Downing M T, Besner G E, Brigstock D R
Department of Surgery, Ohio State University College of Medicine, Columbus 43205, USA.
J Pediatr Surg. 1995 Dec;30(12):1668-72. doi: 10.1016/0022-3468(95)90448-4.
C1300 is a murine neuroblastoma that arose spontaneously in an A/JAX mouse, and from which a clone termed TBJ was subsequently derived. C1300 is a slowly growing and poorly metastasizing tumor, whereas TBJ shows early systemic metastasis as well as aggressive local growth. Compared with TBJ cells, C1300 cells are highly immunogenic and are sensitive to natural killer cells and cytotoxic lymphocytes. In vitro, TBJ cells were found to be more rounded and less adherent than C1300 cells. Because the underlying basis for the differences between C1300 and TBJ cells has not been fully elucidated, the authors used high-resolution two-dimensional gel electrophoresis (2-DE) to study comparative aspects of total protein expression by each cell line. Of the approximately 400 individual cellular proteins that could be resolved using this technique, two were found to be reproducibly and uniquely expressed by TBJ cells and not by C1300 cells. Both proteins were anionic (pl 5.0 to 5.2) as assessed by iso-electric focusing and had molecular weights of 76,000 and 82,000 as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Silver staining of SDS-polyacrylamide gels showed that the levels of 82,000-M(r) protein (p82) were higher than those of the 76,000-M(r) protein (p76). A purification protocol allowing for the isolation of p82 from TBJ cell extracts was developed, which comprised preparative two-dimensional gel electrophoresis followed by reverse-phase high-performance liquid chromatography. Full molecular identification of p82 and p76 eventually may provide new leads in the study of the metastatic or antigenic properties of neuroblastoma.
C1300是一种在A/JAX小鼠中自发产生的鼠神经母细胞瘤，随后从中衍生出一个名为TBJ的克隆。C1300是一种生长缓慢且转移能力较弱的肿瘤，而TBJ则表现出早期全身转移以及侵袭性局部生长。与TBJ细胞相比，C1300细胞具有高度免疫原性，对自然杀伤细胞和细胞毒性淋巴细胞敏感。在体外，发现TBJ细胞比C1300细胞更圆且贴壁性更低。由于C1300和TBJ细胞之间差异的潜在基础尚未完全阐明，作者使用高分辨率二维凝胶电泳（2-DE）来研究每个细胞系总蛋白表达的比较情况。使用该技术可分辨出约400种个体细胞蛋白，其中发现有两种蛋白可重复且独特地由TBJ细胞表达，而C1300细胞不表达。通过等电聚焦评估，这两种蛋白均为阴离子型（等电点5.0至5.2），通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）评估，分子量分别为76,000和82,000。SDS-聚丙烯酰胺凝胶的银染显示，82,000-M（r）蛋白（p82）的水平高于76,000-M（r）蛋白（p76）。开发了一种从TBJ细胞提取物中分离p82的纯化方案，该方案包括制备性二维凝胶电泳，随后进行反相高效液相色谱。p82和p76的完整分子鉴定最终可能为神经母细胞瘤转移或抗原特性的研究提供新线索。
