Factors influencing the promotion of transformation in chemically-initiated C3H/10T1/2 Cl 8 mouse embryo fibroblasts.
Factors influencing the promotion of transformation in chemically-initiated C3H/10T1/2 Cl 8 mouse embryo fibroblasts.
复制标题
影响化学引发的 C3H/10T1/2 Cl 8 小鼠胚胎成纤维细胞转化促进的因素。
作者：
J. H. Frazelle;D. Abernethy;C. Boreiko
用N-甲基-N‘-硝基-N-亚硝基-N-亚硝胺(MNNG)处理C3H/10T1/2Cl8低密度非同步培养的小鼠胚胎成纤维细胞，只有当处理后的细胞暴露于肿瘤促进剂12-O-十四酰佛波醇-13-乙酸酯(TPA)时，才会启动转化过程并产生大量转化灶。研究了影响该启动和促进系统结果的细胞培养变量。发现促进病灶产生的最佳TPA浓度为0.25微克/毫升，在对数生长和整个融合过程中都需要TPA的存在。大量用于培养细胞的胎牛血清在病灶的产生中也起着决定性的作用。在从四个不同供应商购买的九批血清中，只有两批适合MNNG和TPA的启动和推广研究。相比之下，这些批次中有7批足以用于3-甲基胆蒽的转化研究。对Focus生产产生不利影响的因素包括使用杀菌灵或使用高传代菌种培养。这些研究表明，影响这些细胞中促进作用的细胞培养变量是可以控制的，该系统可以成功地用于体外促进作用的细胞机制的研究和遗传毒性物质的检测。
Treatment of low density asynchronous cultures of C3H/10T1/2 Cl 8 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiates the process of transformation and produces significant numbers of transformed foci only when treated cultures are subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell culture variables which influence the outcome of this initiation and promotion system were studied. A TPA concentration of 0.25 micrograms/ml was found to be optimal for the promotion of focus production and the presence of TPA was required both during logarithmic growth and throughout confluence. The lot of fetal calf serum used to cultivate the cells also played a determining role in focus production. Of nine serum lots purchased from four different suppliers, only two were suited for initiation and promotion studies with MNNG and TPA. In contrast, seven of these lots were adequate for transformation studies with 3-methylcholanthrene. Factors which adversely influenced focus production included the use of fungizone or the use of high passage stock cultures. These studies demonstrate that cell culture variables which influence promotion in these cells can be controlled and that this system can be successfully used in studies of the cellular mechanism of in vitro promotion and for the detection of genotoxic substances.