恶性转化人支气管上皮细胞基因组甲基化观察

目的对反式二氢二醇环氧苯并芘（anti-BPDE）和结晶型硫化镍（NiS）恶性转化人支气管上皮细胞（Hu-man bronchial epithelia,16HBE）基因组DNA甲基化状况进行研究,寻找DNA甲基化异常的基因片段,探讨反式-BPDE和NiS的表遗传致癌机制.方法采用限制性指纹识别技术（MSRF）,对反式-BPDE和NiS分别诱导转化及裸鼠成瘤的4种人支气管上皮细胞株基因组进行分析;对异常甲基化基因阳性片断采用TA克隆技术构建测序载体,对测序结果进行同源性分析比较.结果发现结晶型NiS恶性转化人支气管上皮细胞基因组存在高甲基化的DNA片段,其中一基因片段与编码鼻咽癌易感性蛋白ANKRD11基因序列99%同源.另一基因片段与HOXA3基因序列99%同源;未发现反式BPDE恶性转化人支气管上皮细胞基因组DNA异常甲基化基因片段.结论结晶型NiS恶性转化人支气管上皮细胞DNA的高度甲基化可能导致基因表达抑制.可能是结晶型NiS致癌的一种表遗传机制;反式BPDE致癌过程可能与基因组磷酸胞苷酰（CpG）岛甲基化异常关系不明确.

Identification of aberrant DNA methylation in transformed human bronchial epithelia

Objective To study aberrant DNA methylation potentially resulting in the changes of the anti-7,8,-dihydrodiol-9,10-epoxide benzo[a]pyrene(anti-BPDE)and nickel sulfide(NiS)transformed Human bronchial epithelia(16 HBE)as a possible epigenetic mechanism for carcinogenesis.Methods The product DNA isolated from five kinds of 16HBE was analyzed for aberrant methylation using PCR based technique-methylation sensitive restriction fingerprinting technique(MSRF).Several DNA fragments differentially methylated in the transformed cells found by MSRF were ligated to pMD18T Vector and transformed into bacteria.The plasmid DNA were sequenced and compared with data in GenBank by BLASTN.Results DNA hypermethylation was indentified in the NiS transformed 16 HBE,and it was found that one of the DNA fragments homologized(99%)with ANKRD11 which encoded the susceptibility protein of nanopbaryngeal carcinoma and another homologized(99%)with bomeo box A3.Conclusion Anti-BPDE induced 16HBE did not show aberrant genome CpG methylation,which suggestes that aberrant methylation of genes may not happen for anti-BPDE-induced cells transformation and carcinogenesis.DNA hypermethylation is known to result in gene silencing,and it appears that hypermethylation of gene may represent a possible epigenetic mechanism for NiS induced cells transformation and carcinogenesis.