Shibui S, Matsuoka K, Nomura K, Iwasaki K, Hoshino T, Jatreboff M M
Dept. of Neurological Surgery, School of Medicine, University of California, San Francisco 94143.
Gan To Kagaku Ryoho. 1988 Jan;15(1):135-9.
Thymidylate synthase was identified at the cellular level using anti-thymidylate synthase monoclonal antibody (M-TS-4) developed against HeLa cell line. HeLa cells, 9L rat gliosarcoma cells, and some of human brain tumor cells (medulloblastoma, metastatic brain tumors from lung cancer and osteosarcoma) were cultured in complete medium for 72 hr and fixed with 10% buffered formalin. These were covered with 1:20 dilution of M-TS-4 in Burridge buffer and 1% bovine serum albumin for 4 or 24 hr. After rinsing twice with phosphate-buffered saline solution (PBS), the cell staining was made with avidin-biotin peroxidase complex (ABC). In addition, HeLa cells were exposed to 2 microCi/ml of tritiated thymidine for 30 min, cultured again for 0 to 5 hr, and subjected to autoradiography after M-TS-4 staining with ABC. All cells were stained satisfactorily with ABC except 9L rat gliosarcoma cells. Autoradiography revealed that 38% of the cells were stained with ABC, 28% were labeled with tritiated thymidine, while only 8% of the cells were stained simultaneously at 0 hr specimen. However, the cells labeled with both agents subsided when the cells were incubated in complete medium for 1 or 2 hr before fixation. Therefore, thymidylate synthase appears to exist mainly in G1-phase and to subside in early S-phase. Although the number of thymidylate synthase positive cells was greater than that of the cells labeled with tritiated thymidine, the ratio was constant (r = 0.99). The fraction of S-phase can be estimated from that of thymidylate synthase positive cells. Thymidylate synthase positive cell fraction may become another important segment for cell cycle analysis.
使用针对HeLa细胞系开发的抗胸苷酸合成酶单克隆抗体（M-TS-4）在细胞水平鉴定胸苷酸合成酶。将HeLa细胞、9L大鼠胶质瘤细胞和一些人脑肿瘤细胞（髓母细胞瘤、肺癌和骨肉瘤的脑转移瘤）在完全培养基中培养72小时，并用10%缓冲福尔马林固定。用Burridge缓冲液和1%牛血清白蛋白将M-TS-4稀释至1:20覆盖这些细胞4或24小时。用磷酸盐缓冲盐水溶液（PBS）冲洗两次后，用抗生物素蛋白-生物素过氧化物酶复合物（ABC）进行细胞染色。此外，将HeLa细胞暴露于2微居里/毫升的氚标记胸腺嘧啶核苷中30分钟，再次培养0至5小时，并在用ABC进行M-TS-4染色后进行放射自显影。除9L大鼠胶质瘤细胞外，所有细胞用ABC染色效果良好。放射自显影显示，38%的细胞用ABC染色，28%的细胞用氚标记胸腺嘧啶核苷标记，而在0小时标本时只有8%的细胞同时被染色。然而，在固定前将细胞在完全培养基中孵育1或2小时后，两种试剂标记的细胞数量减少。因此，胸苷酸合成酶似乎主要存在于G1期，并在S期早期减少。虽然胸苷酸合成酶阳性细胞的数量大于用氚标记胸腺嘧啶核苷标记的细胞数量，但比例是恒定的（r = 0.99）。S期的比例可以从胸苷酸合成酶阳性细胞的比例中估计出来。胸苷酸合成酶阳性细胞比例可能成为细胞周期分析的另一个重要部分。
