人类恶性畸胎瘤PA-1细胞的p53基因结构、功能状态及其意义

目的:探讨人类恶性畸胎瘤PA-1细胞株p53基因的结构、功能状态及其意义。方法:采用DNA碱基序列分析、FASAY（酵母细胞中单个等位基因的功能分析）及Western blot（蛋白印迹分析）等方法对经20余年407-445继代培养的PA-1细胞株p53的基因结构、功能状态进行了研究。结果:RT-PCR产物DNA定向序列分析显示具有野生及突变2个带（p53密码子239突变）,FASAY结果也显示p53一个等位基因是有活性的（野生的）,而另一个是非活性的（突变的）。Western blot未检测出突变的p53基因蛋白,而p21蛋白的表达水平比正常成纤维细胞低。结论:人卵巢恶性畸胎瘤PA-1细胞株经20年的继代培养后,一个p53等位基因发生错义突变,另一个仍然是野生型的。仅一个p53等位基因发生突变,不足以引起细胞的染色体不稳定性。因此,研究PA-1细胞稳定核型的维持结构,在确定有关染色体的稳定性与不稳定性的基因的方面是非常重要的。

Study on p53 gene construction and functional status of PA-1 cells derived from human teratocarcinoma

Objective:To understand the construction,functional status and the meaning of p53 gene of PA-1 cells derived from human teratocarcinoma.Methods:DNA basic sequence analysis,FASAY(functional and sequencing analysis of separated alleles in yeast)and Western blot analysis were used.Results:Direct sequence analysis RT-PCR products showed both wild and mutated bands(p53 colon 239 mutation), FASAY showed that one allele of the p53 gene was active(wild) .while the other was inactive( mutant) .In Western blot analysis haven't defect the gene protein of p53, while the PA-1 cells expressed the p21 protein to a lesser extent than normal human tibroblaste. Conclusion:One type of the p53 allele gene of PA-1 cells of human teratocarcinoma was detected as missense mutation after cultured for more than twenty years, and this mutation is not sufficient to induce the instabih'ty of the chromosome of cell line. So that the study for the maintenance contruction of the stability nuclein type of PA-1 cells is very important to determin whether the genes is stability or instability of chromosomes.