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目的 研究DNA甲基化与衰老细胞中细胞周期负调控因子p16 MTS1/INK4a高表达的关系。 方法 应用甲基化敏感的DNA限制性内切酶与PCR相结合的方法 ,分析特定位点的甲基化状态。 结果 人胚肺二倍体成纤维细胞衰老过程中p16基因表达增高 ,中年细胞及衰老细胞中p16基因的表达水平分别约为年轻细胞的 3倍和 10倍。p16基因外显子Ⅰ限制性内切酶SmaⅠ位点的甲基化水平则表现出随增龄而降低的趋势 ,在年轻细胞、中年细胞中分别约为 6 4%和 41% ,虽然在衰老细胞中仅为 2 4% ,但仍保持一定的水平。 结论 p16基因外显子Ⅰ的SmaⅠ位点的甲基化水平改变可能与其在衰老过程中的高表达有一定的关联 ,其重要性值得进一步研究。
Objective To study the relationship between DNA methylation and high expression of p16 MTS1 / INK4a, a negative regulator of cell cycle, in senescent cells. Methods Methylation-sensitive DNA restriction endonucleases were combined with PCR to analyze the methylation status of specific sites. Results The expression of p16 gene in human embryo lung diploid fibroblasts was increased during senescence. The expression levels of p16 gene in middle-aged and senescent cells were about 3 and 10 times that of young cells, respectively. The methylation level of SmaI site of exon Ⅰ restriction site of p16 gene showed a trend of decreasing with increasing age, which was about 64% and 41% in young and middle-aged cells, respectively Senescent cells in only 24%, but still maintain a certain level. Conclusion The change of methylation level of SmaⅠ in exon Ⅰ of p16 gene may be related to its high expression in aging process, and its importance is worth further study.