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目的 对镉转化细胞DNA异常甲基化及其对肿瘤相关基因表达的影响进行研究 ,探讨镉的外遗传致癌机制。方法 从CdCl2 转化BALB/c 3T3细胞中提取基因组DNA ,经甲基化非敏感性酶 (Mse1 )单独消化或Mse1和甲基化敏感性酶 (BstU1 )联合消化 ,消化产物用甲基化敏感性内切酶指纹法 (MSRF)进行分析 ,差异显示出异常甲基化基因片段 ,进一步以异常甲基化DNA为探针进行Southern分子杂交加以证实 ,并进行DNA序列测定 ,与基因文库中的基因进行类比分析。结果 发现镉转化细胞存在异常甲基化DNA ,其中一个甲基化DNA片段为p1 6抑癌基因。结论 DNA高甲基化会导致基因表达抑制 ,因此 ,p1 6基因高甲基化会导致其抑癌功能减弱或丧失 ,这可能是镉诱导细胞转化及其致癌作用的一种外遗传机制
Objective To investigate the abnormal DNA methylation of cadmium-transformed cells and its effect on the expression of tumor-related genes in order to explore the mechanism of extra-genetic carcinogenesis of cadmium. Methods Genomic DNA was extracted from CdCl2-transformed BALB / c 3T3 cells and digested with Mse1 or Mst1 and BstU1 respectively. The digestion products were analyzed by methylation-sensitive Endogenous enzyme fingerprinting (MSRF) analysis, the difference shows abnormal methylation gene fragments, and further to abnormal methylated DNA probe for Southern hybridization to be confirmed and DNA sequencing, and gene library genes Perform an analogy analysis. As a result, abnormally methylated DNA was found in the cadmium-transformed cells, in which a methylated DNA fragment was a p16 suppressor gene. CONCLUSION: Hypermethylation of DNA causes inhibition of gene expression. Therefore, hypermethylation of p16 gene may result in its suppression or loss of tumor suppressor function, which may be an epigenetic mechanism of cadmium-induced cell transformation and its carcinogenesis